Superparamagnetic Iron Oxide Nanoparticles Reprogram the Tumor Microenvironment and Reduce Lung Cancer Regrowth after Crizotinib Treatment.

ALK-positive NSCLC patients demonstrate initial responses to ALK tyrosine kinase inhibitor (TKI) treatments, but eventually develop resistance, causing rapid tumor relapse and poor survival rates. Growing evidence suggests that the combination of drug and immune therapies greatly improves patient survival; however, due to the low immunogenicity of the tumors, ALK-positive patients do not respond to currently available immunotherapies. Tumor-associated macrophages (TAMs) play a crucial role in facilitating lung cancer growth by suppressing tumoricidal immune activation and absorbing chemotherapeutics. However, they can also be programmed toward a pro-inflammatory tumor suppressive phenotype, which represents a highly active area of therapy development. Iron loading of TAMs can achieve such reprogramming correlating with an improved prognosis in lung cancer patients. We previously showed that superparamagnetic iron oxide nanoparticles containing core-cross-linked polymer micelles (SPION-CCPMs) target macrophages and stimulate pro-inflammatory activation. Here, we show that SPION-CCPMs stimulate TAMs to secrete reactive nitrogen species and cytokines that exert tumoricidal activity. We further show that SPION-CCPMs reshape the immunosuppressive Eml4-Alk lung tumor microenvironment (TME) toward a cytotoxic profile hallmarked by the recruitment of CD8+ T cells, suggesting a multifactorial benefit of SPION-CCPM application. When intratracheally instilled into lung cancer-bearing mice, SPION-CCPMs delay tumor growth and, after first line therapy with a TKI, halt the regrowth of relapsing tumors. These findings identify SPIONs-CCPMs as an adjuvant therapy, which remodels the TME, resulting in a delay in the appearance of resistant tumors.

Iron homeostasis in mice: does liver lobe matter?

The liver plays a crucial role in maintaining systemic iron homeostasis through iron storage, sensing of systemic iron needs, and production of the iron-regulatory hormone hepcidin. While mice are commonly used as models for studying human iron homeostasis, their liver structure differs significantly from humans. Since the mouse liver is structured in six separated lobes, often, the analysis of a single defined lobe is preferred due to concerns over data reproducibility between experimental cohorts. In this study, we compared iron-related parameters in distinct liver lobes of C57BL/6 wild-type mice across different ages. We found that the non-heme iron levels, as well as the mRNA and protein expression of iron storage protein Ferritin and the iron importer Transferrin Receptor 1, were similar between liver lobes. Additionally, the mRNA expression of Hepcidin, as well as its regulators, Bmp2 and Bmp6, and iron importers Zip8 and Zip14 were comparable. Minor differences were observed in Ferroportin mRNA levels of 24-wk-old mice; however, this did not correlate with altered iron content. The findings in wild-type mice were reproduced in Hfe knock-out mice - a well-established genetic model of the most prevalent form of hemochromatosis. Overall, our results indicate that C57BL/6 mouse liver lobes can be used interchangeably for assessing iron content and expression of iron-related genes. Understanding if these findings are applicable to other mouse developmental stages, strains, or models of (iron-related) disorders will be key to promote reduction of experimental animal numbers and facilitate resource sharing among research groups studying liver iron homeostasis.NEW & NOTEWORTHY This study reveals that, despite being structurally separated, liver lobes from C57BL/6 wild-type and iron-overloaded mice can be used interchangeably for the evaluation of iron content and expression of iron-related genes.

Repression of the iron exporter ferroportin may contribute to hepatocyte iron overload in individuals with type 2 diabetes.

OBJECTIVE: Hyperferremia and hyperferritinemia are observed in patients and disease models of type 2 diabetes mellitus (T2DM). Likewise, patients with genetic iron overload diseases develop diabetes, suggesting a tight link between iron metabolism and diabetes. The liver controls systemic iron homeostasis and is a central organ for T2DM. Here, we investigate how the control of iron metabolism in hepatocytes is affected by T2DM. METHODS: Perls Prussian blue staining was applied to analyze iron distribution in liver biopsies of T2DM patients. To identify molecular mechanisms underlying hepatocyte iron accumulation we established cellular models of insulin resistance by treatment with palmitate and insulin. RESULTS: We show that a subset of T2DM patients accumulates iron in hepatocytes, a finding mirrored in a hepatocyte model of insulin resistance. Iron accumulation can be explained by the repression of the iron exporter ferroportin upon palmitate and/or insulin treatment. While during palmitate treatment the activation of the iron regulatory hormone hepcidin may contribute to reducing ferroportin protein levels in a cell-autonomous manner, insulin treatment decreases ferroportin transcription via the PI3K/AKT and Ras/Raf/MEK/ERK signaling pathways. CONCLUSION: Repression of ferroportin at the transcriptional and post-transcriptional level may contribute to iron accumulation in hepatocytes observed in a subset of patients with T2DM.

The role of iron in chronic inflammatory diseases: from mechanisms to treatment options in anemia of inflammation.

Anemia of inflammation (AI) is a highly prevalent comorbidity in patients affected by chronic inflammatory disorders, such as chronic kidney disease, inflammatory bowel disease, or cancer, that negatively affect disease outcome and quality of life. The pathophysiology of AI is multifactorial, with inflammatory hypoferremia and iron-restricted erythropoiesis playing a major role in the context of disease-specific factors. Here, we review the recent progress in our understanding of the molecular mechanisms contributing to iron dysregulation in AI, the impact of hypoferremia and anemia on the course of the underlying disease, and (novel) therapeutic strategies applied to treat AI.

Iron- and erythropoietin-resistant anemia in a spontaneous breast cancer mouse model.

Anemia of cancer (AoC) with its multifactorial etiology and complex pathology is a poor prognostic indicator for cancer patients. One of the main causes of AoC is cancer-associated inflammation that activates mechanisms, commonly observed in anemia of inflammation, whereby functional iron deficiency and iron-restricted erythropoiesis are induced by increased hepcidin levels in response to raised levels of interleukin-6. So far only a few AoC mouse models have been described, and most of them did not fully recapitulate the interplay of anemia, increased hepcidin levels and functional iron deficiency in human patients. To test if the selection and the complexity of AoC mouse models dictates the pathology or if AoC in mice per se develops independently of iron deficiency, we characterized AoC in Trp53floxWapCre mice that spontaneously develop breast cancer. These mice developed AoC associated with high levels of interleukin-6 and iron deficiency. However, hepcidin levels were not increased and hypoferremia coincided with anemia rather than causing it. Instead, an early shift in the commitment of common myeloid progenitors from the erythroid to the myeloid lineage resulted in increased myelopoiesis and in the excessive production of neutrophils that accumulate in necrotic tumor regions. This process could not be prevented by either iron or erythropoietin treatment. Trp53floxWapCre mice are the first mouse model in which erythropoietin-resistant anemia is described and may serve as a disease model to test therapeutic approaches for a subpopulation of human cancer patients with normal or corrected iron levels who do not respond to erythropoietin.

The Macrophage Iron Signature in Health and Disease.

Throughout life, macrophages are located in every tissue of the body, where their main roles are to phagocytose cellular debris and recycle aging red blood cells. In the tissue niche, they promote homeostasis through trophic, regulatory, and repair functions by responding to internal and external stimuli. This in turn polarizes macrophages into a broad spectrum of functional activation states, also reflected in their iron-regulated gene profile. The fast adaptation to the environment in which they are located helps to maintain tissue homeostasis under physiological conditions.

Core Cross-Linked Polymeric Micelles for Specific Iron Delivery: Inducing Sterile Inflammation in Macrophages.

Iron is an essential co-factor for cellular processes. In the immune system, it can activate macrophages and represents a potential therapeutic for various diseases. To specifically deliver iron to macrophages, iron oxide nanoparticles are embedded in polymeric micelles of reactive polysarcosine-block-poly(S-ethylsulfonyl-l-cysteine). Upon surface functionalization via dihydrolipoic acid, iron oxide cores act as crosslinker themselves and undergo chemoselective disulfide bond formation with the surrounding poly(S-ethylsulfonyl-l-cysteine) block, yielding glutathione-responsive core cross-linked polymeric micelles (CCPMs). When applied to primary murine and human macrophages, these nanoparticles display preferential uptake, sustained intracellular iron release, and induce a strong inflammatory response. This response is also demonstrated in vivo when nanoparticles are intratracheally administered to wild-type C57Bl/6N mice. Most importantly, the controlled release concept to deliver iron oxide in redox-responsive CCPMs induces significantly stronger macrophage activation than any other iron source at identical iron levels (e.g., Feraheme), directing to a new class of immune therapeutics.

The role of cellular iron deficiency in controlling iron export.

BACKGROUND: Iron export via the transport protein ferroportin (Fpn) plays a critical role in the regulation of dietary iron absorption and iron recycling in macrophages. Fpn plasma membrane expression is controlled by the hepatic iron-regulated hormone hepcidin in response to high iron availability and inflammation. Hepcidin binds to the central cavity of the Fpn transporter to block iron export either directly or by inducing Fpn internalization and lysosomal degradation. Here, we investigated whether iron deficiency affects Fpn protein turnover. METHODS: We ectopically expressed Fpn in HeLa cells and used cycloheximide chase experiments to study basal and hepcidin-induced Fpn degradation under extracellular and intracellular iron deficiency. CONCLUSIONS/GENERAL SIGNIFICANCE: We show that iron deficiency does not affect basal Fpn turnover but causes a significant delay in hepcidin-induced degradation when cytosolic iron levels are low. These data have important mechanistic implications supporting the hypothesis that iron export is required for efficient targeting of Fpn by hepcidin. Additionally, we show that Fpn degradation is not involved in protecting cells from intracellular iron deficiency.

Regulation of iron homeostasis: Lessons from mouse models.

Iron is an essential micronutrient and a critical cofactor for proteins involved in fundamental processes such as oxygen transport, energy production and DNA synthesis. However, iron levels need to be tightly balanced to avoid pathological consequences of iron overload or deficiency. Genetically engineered mouse models with alterations in systemic or cellular iron handling advanced our knowledge how systemic and cellular iron homeostasis is maintained. Here, we prepared a comprehensive overview of mouse models that provide insight into mechanisms of iron regulation and/or rare or frequent iron-related disorders.

The hepcidin-ferroportin axis controls the iron content of Salmonella-containing vacuoles in macrophages.

Macrophages release iron into the bloodstream via a membrane-bound iron export protein, ferroportin (FPN). The hepatic iron-regulatory hormone hepcidin controls FPN internalization and degradation in response to bacterial infection. Salmonella typhimurium can invade macrophages and proliferate in the Salmonella-containing vacuole (SCV). Hepcidin is reported to increase the mortality of Salmonella-infected animals by increasing the bacterial load in macrophages. Here we assess the iron levels and find that hepcidin increases iron content in the cytosol but decreases it in the SCV through FPN on the SCV membrane. Loss-of-FPN from the SCV via the action of hepcidin impairs the generation of bactericidal reactive oxygen species (ROS) as the iron content decreases. We conclude that FPN is required to provide sufficient iron to the SCV, where iron serves as a cofactor for the generation of antimicrobial ROS rather than as a nutrient for Salmonella.

Molecular characterization of CD44+/CD24-/Ck+/CD45- cells in benign and malignant breast lesions.

Breast cancer epithelial cells with the CD44+/CD24-/low phenotype possess tumor-initiating cells and epithelial-mesenchymal transition (EMT) capacity. Massive parallel sequencing can be an interesting approach to deepen the molecular characterization of these cells. We characterized CD44+/CD24-/cytokeratin(Ck)+/CD45- cells isolated through flow cytometry from 43 biopsy and 6 mastectomy samples harboring different benign and malignant breast lesions. The Ion Torrent Ampliseq Cancer Hotspot panel v2 (CHPv2) was used for the identification of somatic mutations in the DNA extracted from isolated CD44+/CD24-/Ck+/CD45- cells. E-Cadherin and vimentin immunohistochemistry was performed on sections from the corresponding formalin-fixed, paraffin-embedded (FFPE) blocks. The percentage of CD44+/CD24-/Ck+/CD45- cells increased significantly from non-malignant to malignant lesions and in association with a significant increase in the expression of vimentin. Non-malignant lesions harbored only a single-nucleotide polymorphism (SNP). Mutations in the tumor suppressor p53 (TP53), NOTCH homolog 1 (NOTCH1), phosphatase and tensin homolog (PTEN), and v-akt murine thymoma viral oncogene homolog 1 (AKT1) genes were found in isolated CD44+/CD24-/Ck+/CD45- cells from ductal carcinomas in situ (DCIS). Additional mutations in the colony-stimulating factor 1 receptor (CSF1R), ret proto-oncogene (RET), and TP53 genes were also identified in invasive ductal carcinomas (IDCs). The use of massive parallel sequencing technology for this type of application revealed to be extremely effective even when using small amounts of DNA extracted from a low number of cells. Additional studies are now required using larger cohorts to design an appropriate mutational profile for this phenotype.

HFE Variants and the Expression of Iron-Related Proteins in Breast Cancer-Associated Lymphocytes and Macrophages.

The association of HFE (High Iron FE) major variants with breast cancer risk and behavior has been a matter of discussion for a long time. However, their impact on the expression of iron-related proteins in the breast cancer tissue has never been addressed. In the present study, hepcidin, ferroportin 1, transferrin receptor 1 (TfR1), and ferritin expressions, as well as tissue iron deposition were evaluated in a collection of samples from breast cancers patients and analyzed according to the patients' HFE genotype. Within the group of patients with invasive carcinoma, those carrying the p.Cys282Tyr variant in heterozygosity presented a higher expression of hepcidin in lymphocytes and macrophages than wild-type or p.His63Asp carriers. An increased expression of TfR1 was also observed in all the cell types analyzed but only in p.Cys282Tyr/p.His63Asp compound heterozygous patients. A differential impact of the two HFE variants was further noticed with the observation of a significantly higher percentage of p.Cys282Tyr heterozygous patients presenting tissue iron deposition in comparison to p.His63Asp heterozygous. In the present cohort, no significant associations were found between HFE variants and classical clinicopathological markers of breast cancer behavior and prognosis. Although limited by a low sampling size, our results provide a new possible explanation for the previously reported impact of HFE major variants on breast cancer progression, i.e., not by influencing systemic iron homeostasis but rather by differentially modulating the local cellular expression of iron-related proteins and tissue iron deposition.

Characterization of CD44+ALDH1+Ki-67- Cells in Non-malignant and Neoplastic Lesions of the Breast.

BACKGROUND: Cancer stem cells are tumor cells that present self-renewal, clonal tumor initiation capacity and clonal long-term repopulation potential. We have previously demonstrated that the co-expression of the breast cancer stem cell (BCSC) markers hyaluronan receptor (CD44) and aldehyde dehydrogenase-1 (ALDH1) in ductal carcinomas in situ could be determinant for disease progression. Combining these established BCSC markers with Ki-67 to evaluate quiescence we sought to identify, evaluate the distribution and estimate the mean percentages of CD44(+)ALDH1(+)Ki-67(-) breast cells. MATERIALS AND METHODS: Triple-immunohistochemistry for CD44, ALDH1 and Ki-67 was applied in a series of 16 normal, 54 non-malignant and 155 malignant breast tissues. Clinical relevance was inferred by associations with markers of breast cancer behavior, progression and survival. RESULTS: The mean percentages of cells with this phenotype increased significantly from non-malignant lesions to high-grade ductal carcinomas in situ, decreasing in invasive ductal carcinomas, as also evidenced by an inverse correlation with histological grade and tumor size. The mean percentage of CD44(+)ALDH1(+)Ki-67(-) cells was also significantly higher in women who developed distant metastasis and died due to breast cancer, and a significant association with human epidermal growth factor type 2 (HER2) negativity was observed. CONCLUSION: Our novel findings indicate that CD44(+)ALDH1(+)Ki-67(-) tumor cells may favor distant metastasis and can predict overall survival in patients with ductal carcinomas of the breast. More importantly, quiescence may have a crucial role for tumor progression, treatment resistance and metastatic ability of BCSCs.

Local iron homeostasis in the breast ductal carcinoma microenvironment.

BACKGROUND: While the deregulation of iron homeostasis in breast epithelial cells is acknowledged, iron-related alterations in stromal inflammatory cells from the tumor microenvironment have not been explored. METHODS: Immunohistochemistry for hepcidin, ferroportin 1 (FPN1), transferrin receptor 1 (TFR1) and ferritin (FT) was performed in primary breast tissues and axillary lymph nodes in order to dissect the iron-profiles of epithelial cells, lymphocytes and macrophages. Furthermore, breast carcinoma core biopsies frozen in optimum cutting temperature (OCT) compound were subjected to imaging flow cytometry to confirm FPN1 expression in the cell types previously evaluated and determine its cellular localization. RESULTS: We confirm previous results by showing that breast cancer epithelial cells present an 'iron-utilization phenotype' with an increased expression of hepcidin and TFR1, and decreased expression of FT. On the other hand, lymphocytes and macrophages infiltrating primary tumors and from metastized lymph nodes display an 'iron-donor' phenotype, with increased expression of FPN1 and FT, concomitant with an activation profile reflected by a higher expression of TFR1 and hepcidin. A higher percentage of breast carcinomas, compared to control mastectomy samples, present iron accumulation in stromal inflammatory cells, suggesting that these cells may constitute an effective tissue iron reservoir. Additionally, not only the deregulated expression of iron-related proteins in epithelial cells, but also on lymphocytes and macrophages, are associated with clinicopathological markers of breast cancer poor prognosis, such as negative hormone receptor status and tumor size. CONCLUSIONS: The present results reinforce the importance of analyzing the tumor microenvironment in breast cancer, extending the contribution of immune cells to local iron homeostasis in the tumor microenvironment context.

Co-expression of stem cell markers ALDH1 and CD44 in non-malignant and neoplastic lesions of the breast.

BACKGROUND/AIM: The Cancer Stem Cell (CSC) model proposes that cancer is driven by a cellular component which possesses stem cell (SC) properties, cancer stem cells (CSCs), a distinct cell-type which is tumorigenic and capable of invasion and metastasis. Enzymatic activity of aldehyde dehydrogenase-1 (ALDH1), a de-toxifying enzyme that oxidizes intracellular aldehydes, has been used as a marker of normal and malignant breast stem cells (BSCs). CD44-transmembrane protein has already been shown to possess the ability to identify breast epithelial cells with stem properties. MATERIALS AND METHODS: In order to compare two of the currently most reliable BSCs markers, ALDH1 and CD44, and to correlate their expression within different breast lesions, 190 samples from breast cancer specimens were analyzed by tissue microarrays. RESULTS: ALDH1 expression was observed in 85.43% and CD44 in 90.3% of all samples. No overexpression was observed for ALDH1 between invasive tumors, ductal carcinomas in situ and non-malignant lesions of breast, although ALDH1 had a significant negative correlation with estrogen-receptor (ER) and progesterone-receptor (PR) status (p=0.002 and p=0.001, respectively) and a positive correlation with CD44 (p<0.001). Moreover, combined overexpression of ALDH1 and CD44 was observed in ductal in situ tumors (p<0.001). CONCLUSION: The combined overexpression of these markers in ductal carcinomas in situ is in agreement with the CSC model in breast cancer.

Iron homeostasis in breast cancer.

Iron is an essential element and a critical component of molecules involved in energy production, cell cycle and intermediate metabolism. However, the same characteristic chemistry that makes it so biologically versatile may lead to iron-associated toxicity as a consequence of increased oxidative stress. The fact that free iron accumulates with age and generates ROS led to the hypothesis that it could be involved in the etiogenesis of several chronic diseases. Iron has been consistently linked to carcinogenesis, either through persistent failure in the redox balance or due to its critical role in cellular proliferation. Several reports have given evidence that alterations in the import, export and storage of cellular iron may contribute to breast cancer development, behavior and recurrence. In this review, we summarize the basic mechanisms of systemic and cellular iron regulation and highlight the findings that link their deregulation with breast cancer. To conclude, progresses in iron chelation therapy in breast cancer, as a tool to fight chemotherapy resistance, are also reviewed.