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Soluble pulmonary factors have been reported to be capable of inhibiting the viability of cancer cells that metastasize to the lung, but the molecular identity was obscure. Here we report the isolation and characterization of the beta subunit of hemoglobin as a lung-derived antimetastatic factor. Peptide mapping in the beta subunit of human hemoglobin (HBB) defined a short C-terminal region (termed Metox) as responsible for activity. In tissue culture, both HBB and murine HBB2 mediated growth arrest and apoptosis of lung-metastasizing neuroblastoma cells, along with a variety of other human cancer cell lines. Metox acted similarly and its administration in human tumor xenograft models limited the development of adrenal neuroblastoma tumors as well as spontaneous lung and bone marrow metastases. Expression studies in mice indicated that HBB2 is produced by alveolar epithelial and endothelial cells and is upregulated in mice bearing undetectable metastasis. Our work suggested a novel function for HBB as a theranostic molecule: an innate antimetastasis factor with potential utility as an anticancer drug and a biomarker signaling the presence of clinically undetectable metastasis. Cancer Res; 77(1); 14–26. ©2016 AACR.

This file includes tables presenting quantitative Real-Time PCR oligonucleotide primers and chemically synthesized peptide segments of human HBB. It also contains figures presenting the influence of separated lung-derived factors on neuroblastoma cell viability, the beta subunit of hemoglobin is elevated in micrometastasis-bearing organs of inoculated mice, single channel confocal images of HBB2 expression in alveolar epithelial and endothelial cells, a high expression of HBB2 is seen in alveolar endothelial cells lining blood vessels of varied diameters in micrometastasis-bearing mice, the beta subunit isolated from native human hemoglobin inhibits neuroblastoma cell viability, human HBB and its derivative peptide Metox do not mediate hemolysis of red blood cells, an intravenous or intranasal treatment of Metox inhibits neuroblastoma local tumor growth and metastasis, and nitric oxide is not influenced by high HBB2 levels in the lungs and serum of micrometastasis-bearing mice.

This file includes supplemental experimental procedures. It includes a description of animals, antibodies, dialysis of lung-derived factors, in-vitro viability assays, blocking the inhibitory effect of lung-derived factors, quantitative Real-Time PCR, fixation and sectioning of frozen tissues, immunofluorescence of frozen tissue sections, protein extraction from mouse organs, western blotting, enzyme-Linked Immunosorbent assay, Isolation of primary endothelial and epithelial cells from mouse lungs, Incubation of HPF and HPEC with MicroNB cells in Transwell system, apoptosis assay, cell cycle analysis, ERK1/2, p38 and TAK1 phosphorylation, Solid-phase synthesis of human HBB segments, Conjugation of Metox peptide to FITC, Confocal microscopy for Metox cell entry, Flow cytometry for Metox cell entry, orthotopic inoculation of tumor cells, monitoring tumor growth and metastasis, RNA preparation and Reverse Transcription Polymerase Chain Reaction, quantitative real-time PCR, intravenous and intranasal treatment of Metox, hemolytic assay, and nitric oxide assay.

A solid tumor is an ecosystem composed of tumor cells, resident and infiltrating nontumor cells, and molecules present in proximity to these cells. This ecosystem can be collectively described as the tumor microenvironment. Both the tumor cells as well as the neighboring nontumor cells take part in

CXCL10 was recently shown to exert antimalignancy functions by influencing the tumor microenvironment. Here, we have taken a different approach, investigating the effects of CXCL10 directly on tumor-promoting functions in colorectal carcinoma (CRC) cells. CXCL10 expression was detected in preferred metastatic sites of CRC (liver, lungs, and lymph nodes), and its CXCR3 receptor was expressed by eight CRC cell lines (detected: reverse transcription-PCR and/or flow cytometry). Detailed analysis was done on two cell lines derived from primary CRC tumors (SW480, KM12C) and their metastatic descendents (SW620 and KM12SM). The three known variants of CXCR3 (CXCR3-A, CXCR3-B, and CXCR3-alt) were detected in all four cell lines. CXCR3 expression was also observed on colorectal tumor cells in biopsies of CRC patients (immunohistochemistry). CXCL10 and CXCR3 expression were potently induced in CRC cells by Interferon γ and all four CRC cell lines responded to CXCL10 by extracellular signal-regulated kinase 1/2 dephosphorylation. The chemokine did not affect tumor cell growth or angiogenesis-related functions in the tumor cells, such as CXCL8 and vascular endothelial growth factor secretion. Importantly, CXCL10 significantly up-regulated invasion-related properties in CRC cells: It promoted matrix metalloproteinase 9 expression and induced CRC cell migration. Of note, CXCL10-induced migration was detected only in the two metastatic cells and not in their primary counterparts. Also, CXCL10 promoted the adhesion of metastatic cells to laminin. These results suggest that CXCL10 can be exploited by CRC cells toward their progression, thus possibly antagonizing the antimalignancy effects of the chemokine on the tumor microenvironment. Therefore, care should be taken when considering CXCL10 as a therapeutic antitumor modality for CRC treatment. [Cancer Res 2007;67(7):3396–405]

Extravasation of tumor cells is a pivotal step in metastasis formation. This step is initiated by an interaction of extravasating tumor cells with endothelial cells. Among the molecules mediating tumor-endothelium interactions are selectins and their fucosylated ligands. In a previous study, we demonstrated that the fucose-generating FX enzyme regulates the expression of selectin ligands by B and T lymphocytes and by head and neck squamous cell carcinoma cells. It was also shown that the FX enzyme regulated important interaction parameters between these cancer cells and endothelial cells. The present study was aimed to determine whether the FX enzyme controls adhesive interactions between colorectal cancer cells and endothelial cells. The results clearly indicate that this is indeed the case. Overexpressing the FX enzyme by the transfer of FX cDNA to low FX-expressing colorectal cancer cells resulted in an increased adhesive capacity of the transfectants to activated endothelial cells and to recombinant E-selectin. Down-regulating FX levels in colorectal cancer cells expressing high levels of endogenous FX by transfection with small-interfering RNA resulted in a down-regulated expression of the selectin ligand sialyl Lewis-a and a decrease in the adhesive capacity of the transfectants to activated endothelial cells and to recombinant E-selectin. These transfection experiments also indicated that manipulating the levels of the FX enzyme affected global cellular fucosylation and altered the interaction of colorectal cancer cells with some extracellular matrix components such as fibronectin. We also found that highly metastatic colorectal cancer variants express higher levels of FX and of sialyl Lewis-a than low metastatic variants originating in the same tumors. These results lead us to hypothesize that the FX enzyme controls the capacity of colorectal cancer to extravasate and form metastasis. If this hypothesis will be confirmed the FX enzyme could become a target molecule for metastasis prevention.