Macrophage-tumor cell fusions from peripheral blood of melanoma patients.
BACKGROUND
While the morbidity and mortality from cancer are largely attributable to its metastatic dissemination, the integral features of the cascade are not well understood. The widely accepted hypothesis is that the primary tumor microenvironment induces the epithelial-to-mesenchymal transition in cancer cells, facilitating their escape into the bloodstream, possibly accompanied by cancer stem cells. An alternative theory for metastasis involves fusion of macrophages with tumor cells (MTFs). Here we culture and characterize apparent MTFs from blood of melanoma patients.


METHODS
We isolated enriched CTC populations from peripheral blood samples from melanoma patients, and cultured them. We interrogated these cultured cells for characteristic BRAF mutations, and used confocal microscopy for immunophenotyping, motility, DNA content and chromatin texture analyses, and then conducted xenograft studies using nude mice.


RESULTS
Morphologically, the cultured MTFs were generally large with many pseudopod extensions and lamellipodia. Ultrastructurally, the cultured MTFs appeared to be macrophages. They were rich in mitochondria and lysosomes, as well as apparent melanosomes. The cultured MTF populations were all heterogeneous with regard to DNA content, containing aneuploid and/or high-ploidy cells, and they typically showed large sheets (and/or clumps) of cytoplasmic chromatin. This cytoplasmic DNA was found within heterogeneously-sized autophagic vacuoles, which prominently contained chromatin and micronuclei. Cultured MTFs uniformly expressed pan-macrophage markers (CD14, CD68) and macrophage markers indicative of M2 polarization (CD163, CD204, CD206). They also expressed melanocyte-specific markers (ALCAM, MLANA), epithelial biomarkers (KRT, EpCAM), as well as the pro-carcinogenic cytokine MIF along with functionally related stem cell markers (CXCR4, CD44). MTF cultures from individual patients (5 of 8) contained melanoma-specific BRAF activating mutations. Chromatin texture analysis of deconvoluted images showed condensed DNA (DAPI-intense) regions similar to focal regions described in stem cell fusions. MTFs were readily apparent in vivo in all human melanomas examined, often exhibiting even higher DNA content than the cultured MTFs. When cultured MTFs were transplanted subcutaneously in nude mice, they disseminated and produced metastatic lesions at distant sites.
Apparent MTFs are present in peripheral blood of patients with cutaneous melanomas, and they possess the ability to form metastatic lesions when transplanted into mice. We hypothesize that these MTFs arise at the periphery of primary tumors in vivo, that they readily enter the bloodstream and invade distant tissues, secreting cytokines (such as MIF) to prepare "niches" for colonization by metastasis initiating cells.
Department of Pathology and Gittlen Cancer Research Laboratories, Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania, United States of America.
Gary A
GA
Clawson
Department of Biochemistry & Molecular Biology, Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania, United States of America.
Gail L
GL
Matters
Department of Pathology and Gittlen Cancer Research Laboratories, Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania, United States of America.
Ping
P
Xin
Department of Pharmacology and the Institute for Personalized Medicine, Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania, United States of America.
Yuka
Y
Imamura-Kawasawa
Department of Pathology and Gittlen Cancer Research Laboratories, Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania, United States of America.
Zhen
Z
Du
Department of Dermatology, Division of Health Science Research, Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania, United States of America.
Diane M
DM
Thiboutot
Department of Dermatopathology, Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania, United States of America.
Klaus F
KF
Helm
Department of Surgery and the Melanoma Center, Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania, United States of America.
Rogerio I
RI
Neves
Department of Neural and Behavioral Science and the Microscopy Imaging Facility, Hershey Medical Center, Pennsylvania State University, Hershey, Pennsylvania, United States of America.
Thomas
T
Abraham
D000818:Animals; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor / Q000235:genetics; D015815:Cell Adhesion Molecules; D002459:Cell Fusion; D002843:Chromatin / Q000473:pathology; D000071858:Epithelial Cell Adhesion Molecule; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics*; D006801:Humans; D008264:Macrophages / Q000378:metabolism / Q000473:pathology*; D008544:Melanocytes / Q000378:metabolism / Q000473:pathology; D008545:Melanoma / Q000235:genetics / Q000473:pathology*; D051379:Mice; D018613:Microscopy, Confocal; D009363:Neoplasm Proteins; D014411:Neoplastic Stem Cells / Q000378:metabolism / Q000473:pathology*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D059016:Tumor Microenvironment / Q000235:genetics; D023041:Xenograft Model Antitumor Assays
D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D015815:Cell Adhesion Molecules; D002843:Chromatin; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule; D009363:Neoplasm Proteins; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf