Abstract Here we describe isolation and characterization of macrophage-tumor cell fusions (MTFs) from the blood of pancreatic ductal adenocarcinoma (PDAC) patients. The MTFs were generally aneuploidy, and immunophenotypic characterizations showed that the MTFs express markers characteristic of PDAC and stem cells, as well as M2-polarized macrophages. Single cell RNASeq analyses showed that the MTFs express many transcripts implicated in cancer progression, LINE1 retrotransposons, and very high levels of several long non-coding transcripts involved in metastasis (such as MALAT1). When cultured MTFs were transplanted orthotopically into mouse pancreas, they grew as obvious well-differentiated islands of cells, but they also disseminated widely throughout multiple tissues in “stealth” fashion. They were found distributed throughout multiple organs at 4, 8, or 12 weeks after transplantation (including liver, spleen, lung), occurring as single cells or small groups of cells, without formation of obvious tumors or any apparent progression over the 4 to 12 week period. We suggest that MTFs form continually during PDAC development, and that they disseminate early in cancer progression, forming “niches” at distant sites for subsequent colonization by metastasis-initiating cells. Introduction Pancreatic ductal adenocarcinoma (PDAC) is one of the most prevalent cancers worldwide, and is predicted to be the 2^nd leading cause of cancer deaths by 2030 [[52]1]. PDAC is generally diagnosed at an advanced stage due to lack of early symptoms, precluding surgical excision, and there are no effective alternative treatments. As with most carcinomas, mortality is due to metastatic dissemination, and CTCs are observed in a high proportion of PDAC patients at all stages [[53]2, [54]3]. While there are a number of models for what is termed the “metastatic cascade” [[55]4], the nature of the CTCs which actually produce metastatic foci is not clear. Perhaps the most widely accepted hypothesis underlying metastasis is that the primary tumor microenvironment (TME) induces an epithelial-to-mesenchymal transition (EMT) in a subset of epithelial cancer cells, that facilitates their escape into the bloodstream or lymphatics [[56]5]. A number of studies for example have documented EMT-related changes (and loss of EpCAM expression) in CTCs [[57]6–[58]10]. In spite of recognized shortcomings [[59]11, [60]12], CellSearch quantitation of numbers of EpCAM+ CTCs in peripheral blood has prognostic significance [[61]13–[62]15]. However, the picture remains incomplete: Which CTCs are the capable of initiating metastatic lesions (so called metastasis initiating cells, MICs), and how do MICs find suitable sites for growth of metastatic foci [[63]5]? With regard to the former, a corollary idea is that the EMT-altered cancer cells at the periphery of a primary tumor facilitate liberation of cancer stem cells [[64]5, [65]16, [66]17], which could represent the MICs. In this scenario, the overall number of CTCs would stochastically represent a much smaller subset of MICs. However, this story does not address the latter question: how MICs find suitable “niches” which allow them to establish metastases and proliferate [[67]18]. An alternative theory for metastasis [[68]19–[69]22] involves fusion of macrophages with tumor cells (macrophage-tumor cell fusions, MTFs). With some sort of sorting, recombination, and/or reprogramming [[70]23] of genetic material, this could produce neoplastic cells which have acquired the highly invasive phenotype of macrophages. There is considerable support for this notion from animal models, and some recent support from reports of human cancers [[71]20], but how frequently this occurs is unknown and the basic premise seems to be at odds with the EMT/stem cell hypothesis [[72]18]. We recently reported on MTFs cultured from blood from patients with early-stage and advanced melanomas [[73]24]. The MTFs expressed multiple markers characteristic of M2-polarized macrophages, as well as epithelial, melanocytic and stem cell markers. When the melanoma MTFs were transplanted into mice as subcutaneous xenographs, they disseminated only to pancreas, where they formed what appeared to be benign islands of well-differentiated cells. Here we report on analogous MTFs cultured from blood of PDAC patients. These cells show expression of a similar combination of macrophage and epithelial/pancreatic/stem cell markers. Ultrastructural analyses revealed a macrophage-like morphology, with extensive autophagic vacuoles, etc. Single cell RNASeq analyses showed high levels of expression of various metastasis-related markers (particularly the MIF/CD44/CD74/CXCR4 signaling axis), as well as LINE-1 retrotransposons. In addition, the MTFs uniformly expressed very high levels of MALAT1, a long non-coding RNA transcript known to be involved in control of metastasis [[74]25, [75]26], as well as additional long non-coding transcripts implicated in cancer progression. When the cultured PDAC MTFs were orthotopically transplanted into the pancreas in mice, they formed well-differentiated islands there. They did not form obvious tumors in any other distant locations. However, they were found to disseminate widely throughout multiple tissues, including liver, spleen, lung, submucosa, etc. They were found as single cells or small groups of cells and often appeared large and irregularly shaped. There was no apparent progression in number of cells in various tissues over the 4 to12 week period, although the only metastatic cells found in lung were observed at 12 weeks. The MTFs also appeared to alter their expression of some markers after dissemination. Results Immunophenotypic analysis of cultured MTFs Blood samples were obtained under an approved IRB protocol from patients with PDAC (some patients had early stage resectable disease, although most had advanced disease). Samples were processed as described, and cultured in standard media for ~4–6 weeks. Cells were quite sparse at the outset of culturing (perhaps a few cells/ml). Populations of cells grew from all preparations (~ 20), and they were fixed and stained for various pairs of markers and examined using confocal microscopy, including macrophage markers, pancreatic, epithelial, and pancreatic stem cell markers. The cell populations showed uniform expression (and localization) of the various pairs of human markers (Figs [76]1 and [77]2). Fig 1. Immunostaining of cultured MTFs from PDAC patients. [78]Fig 1 [79]Open in a new tab Representative confocal images of cultured MTFs from different PDAC patients. The cell populations showed uniform staining, and subcellular localization, of the various pairs of markers. Since the plates were sparsely populated, low power panoramic photomicrographs did not allow adequate visualization of the subcellular localizations, so photomicropraphs of individual cells were generally taken. Nuclei were stained with DAPI (Blue) shown in Panels [A, E, I, M, Q and U]. The same cells were also stained for various markers specific for pancreas, PDAC stem cell, or macrophage markers. Panels [B, C, F and G]: PDAC stem cell marker ALDH1A1 (Green) and pancreas marker ZG16B (red). Panels [J, K, N and O]: PDAC stem cell marker ALDH1A1 (Green) and pan-macrophage marker CD68 (Red). Panels [R, S]: Pancreas marker S100PBP (Green) and PDAC stem cell marker CD44 (Red). Panels [V, W]: M2-polarization macrophage marker CD206 (Green) and PDAC stem cell marker CD44 (Red). Composite images are shown in Panels [D, H, L, P, T and X]. Fig 2. Immunostaining of cultured MTFs from PDAC patients. [80]Fig 2 [81]Open in a new tab Representative confocal images of cultured MTFs from different PDAC patients. Nuclei were stained with DAPI (Blue) shown in Panels [A1, E1, I1, M1 and Q1]. The same cells were also stained with various fluorescent markers specific for pancreas, macrophage, the pre-carcinogenic cytokine MIF and its receptor CXCR4. Panel [B1, C1]: MIF (Green) and pancreas-specific marker ZG16B (red). Nuclei had “tunnels” which were stained strongly for MIF. Panels [F1, G1, J1 and K1]: CXCR4 (Green) and pancreas-specific marker ZG16B (red). Panels [N1, O1, R1 and S1]: CXCR4 (Green) and M2-polarization macrophage marker CD204 (Red). Composite images are shown in Panels [D1, H1, L1, P1 and T1]. We also examined cultured MTFs for expression of the pro-carcinogenic cytokine MIF, because of MIF’ s prominent roles in M2 polarization of macrophages, the tumor microenvironment (TME), and cancer progression [[82]27–[83]31]. The cultured PDAC MTFs routinely stained positively for the MIF ([84]Fig 2), with some very intriguing patterns of staining noted. Many of the individual nuclei appeared to have “tunnels” through them. These tunnels (invaginations) were lined by an intact nuclear envelope, and often contained cytoplasmic organelles including mitochondria, etc. (see below). The interior (cytoplasm) within these tunnels stained strongly for MIF, as determined using 3D confocal images (for example, see Panels A1 and B1 of [85]Fig 2). Such tunnels had previously been observed in MTFs cultured from melanoma patient samples, as well as within melanomas in situ [[86]24], and they are also evident in human PDACs (see below). Given the robust immunostaining for MIF, we also examined the functionally related stem cell markers CXCR4 and CD44. CXCR4 is a non-cognate receptor for MIF [[87]32, [88]33] and CD44 represents the signaling component of the MIF:CD74 receptor complex [[89]34]. As with MIF, we observed strong expression of CXCR4 and CD44, indicative of pro-carcinogenic activities of the MIF/CD44/CD74/CXCR4 signaling pathway ([90]Fig 2; see Discussion). Immunophenotypic analysis of primary human PDACs We observed analogous results in tissue specimens from primary human PDACs ([91]Fig 3, Panels A-X). While there was morphologic heterogeneity in various regions of the PDACs, there was a surprisingly extensive subpopulation of cells that dually stained for various pairs of macrophage, pancreatic-epithelial, and stem cell markers ([92]Fig 3). Invaginations (tunnels) were often evident in nuclei of the dual-staining cells (as was also apparent with DAPI-stained PDACs). With 3D confocal images, we also observed dual-staining of these cells for combinations of macrophage markers (CD204 or CD206) and pancreatic cancer markers ZG16B or S100PBP ([93]Fig 4). We also conducted ploidy analysis of the apparent MTFs within PDACs in situ. We found that this population of dual-staining cells contained markedly abnormal DNA contents, with a large portion of the cells showing very irregular nuclei with aneuploid DNA content ([94]Fig 5). These results are similar to those we previously described for melanoma-derived MTFs [[95]24]. Fig 3. Representative confocal images of MTFs in PDAC tissues from different patients. [96]Fig 3 [97]Open in a new tab Nuclei were stained with DAPI (Blue), shown in Panels [A, E, I, M, Q and U]. The same cells were also stained with various markers specific for pancreas, macrophage, or epithelial differentiation. Panels [B, C, F and G]: Epithelial marker pan-cytokeratin (Green) and M2-polarization macrophage marker CD206 (Red). Panels [J, K]: Epithelial marker pan-cytokeratin (Green) and M2-polarization macrophage marker CD163 (Red). Panels [N, O]: Epithelial marker pan-cytokeratin (Green) and M2-polarization macrophage marker CD204 (Red). Panels [R, S]: M2-polarization macrophage marker CD206 (Green) and epithelial marker EpCAM (Red). Panels [V, W]: M2-polarization macrophage marker CD206 (Green) and pancreas-specific marker ZG16B (Red). Composite images are shown in Panels [D, H, L, P, T and X]. Fig 4. Representative 3D confocal images of MTFs in PDAC tissues from different patients which show various nuclear geometries (Yellow) of dual-stained cells. [98]Fig 4 [99]Open in a new tab [Panels A—I]: Dual stained for pancreatic tumor marker ZG16B (Red) and macrophage marker CD 206 (Green); [Panels J & K]: Dual stained for pancreatic tumor marker ZG16B (Red) and macrophage marker CD 204 (Green); [Panel L]: Dual stained for pancreatic tumor marker S100BPB (Green) and macrophage marker CD 204 (Red). Fig 5. Ploidy analysis of dual-staining MTFs in PDACs. [100]Fig 5 [101]Open in a new tab DNA content analysis of dual-staining cells was performed as described in PDACs in archival FFPE tissues. DNA content was also analyzed in adjacent “normal” pancreas (gray bars). Populations of MTFs from 2 patients, showed cells with DNA distribution peaks corresponding to “para-diploid” but with many aneuploidy cells distributed throughout the range, including some with DNA contents ranging up to 5n (black bars). Ultrastructural features of cultured MTFs Ultrastructural examination of the MTFs via transmission electron microscopy (TEM) showed features characteristic of macrophages ([102]Fig 6). The MTFs were generally large cells, which showed exuberant pseudopods, lamellipodia, and exocytosis. Nuclei generally showed very irregular (in fact, jagged) contours, as noted previously with melanoma MTFs [[103]24]. Nuclei of the PDAC MTFs often showed “tunnels” through them (from ultrastructural examination, the concentrated staining for MIF is actually “extranuclear”, within the tunnels or invaginations of cytoplasm). MTFs contained large numbers of mitochondria, lysosomes, autophagic vacuoles, and various autolysomal breakdown products (including laminated bodies structurally comparable to lysosomes), and various structural remnants ([104]Fig 6, Panels G & H). Heterogeneously-sized autophagic vacuoles containing chromatin (and micronuclei) were often a prominent feature, with some very dense remnant bodies ([105]Fig 6H). Fig 6. Transmission electron microscopy of cultured MTFs. [106]Fig 6 [107]Open in a new tab Panels A-H show representative cells with distinctive features, including cytoplasmic “tunnels” through nuclei (Panel G). Focal hyper-dense regions of chromatin (Panel A, E, and F), autophagic bodies (Panel D), etc. are evident within the individual cells. Notably, most nuclei also showed focal areas of condensed chromatin by TEM ([108]Fig 6). They characteristically did not show fibrillar centers, or dense fibrillar or granular components generally seen in nucleoli. These regions may represent the ultrastructural correlate of focal areas of condensed “DAPI-intense” chromatin regions which have been reported in fusions between embryonic stem cells and somatic cells [[109]35], and linked to malignancy in prostate cancer [[110]36]. Single cell RNASeq analyses of cultured MTFs We also performed single cell RNA-Seq expression analyses on cells from 4 patients that both included and excluded assembled transcripts without coding potential (see [111]Materials and Methods for details). Most of the transcripts identified were found in both analyses, although there were a few transcripts of interest which showed up differentially. These notably included CD74 (in the signaling pathway for MIF), which was one of the most highly expressed transcripts across all cells in all patients, as well as a few other genes such as HNRNPA2B1 (see below). Expression clustering was done using complete linkage clustering with Euclidean distance across individual cells. The analysis that excluded transcripts without coding potential identified 5 clusters, which were common to all 4 patient datasets. Cluster 5 contained only 3 transcripts, all representing Long Interspersed Element-1 (LINE-1) retrotransposons (see below), which curiously encoded only ORF2. However, Cluster 1 also contained 14 distinct LINE1 transcripts, encoding both ORF1 and ORF2, as well as HNRNPA2B1, which is an HNRNP component which positively regulates LINE-1 retrotransposition (see Discussion). LINE-1 retrotransposons form the only autonomously active family of transposable elements [[112]37]. Since the RNA transcripts contained both ORF1 and ORF2, this would appear to indicate that this family is actively engaged in moving DNA elements in these cells. Cluster 4 contained only 3 transcripts, composed of ferritin light chain (FLT) and ferritin heavy chain (FTH1), which have been associated with several cancers [[113]38–[114]40]. Ferritin has been found in stroma and tumor-associated macrophages in breast cancer [[115]41], where it was associated with increasing grade and stimulated proliferation of breast cancer cells via an iron-independent mechanism. FTH1 also serves as a novel marker for macrophages [[116]42], and FTL is a prognostic marker in tumor-associated macrophages [[117]43], along with CD163. In the analysis which included non-coding transcripts, FTL and FTH1 were present in cluster 2 (see [118]Table 1). Table 1. Cancer-related genes of interest. Cluster 1 Cluster 2 Cluster 3 Gene Name Reference(s) Gene Name References Gene Name Reference(s)