Ex vivo expansion of lung cancer-derived disseminated cancer cells from lymph nodes identifies cells associated with metastatic progression.

The cellular basis of the apparent aggressiveness in lung cancer is poorly understood but likely associated with functional or molecular features of disseminated cancer cells (DCCs). DCCs from epithelial cancers are mostly detected by antibodies directed against histogenetic markers such as cytokeratin or EpCAM. It has been argued that marker-negative metastatic founder cells might escape detection. We therefore used ex vivo sphere formation for functional detection of candidate metastasis founders. We generated cell suspensions from 199 LN samples of 131 lung cancer patients and placed them into non-adherent cell culture. Sphere formation was associated with detection of DCCs using EpCAM immunocytology and with significantly poorer prognosis. The prognostic impact of sphere formation was strongly associated with high numbers of EpCAM-positive DCCs and aberrant genotypes of expanded spheres. We also noted sphere formation in patients with no evidence of lymphatic spread, however such spheres showed infrequent expression of signature genes associated with spheres from EpCAM-positive samples and displayed neither typical lung cancer mutations (KRAS, TP53, ERBB1) nor copy number variations, but might be linked to disease progression >5 years post curative surgery. We conclude that EpCAM identifies relevant disease-driving DCCs, that such cells can be expanded for model generation and that further research is needed to clarify the functional and prognostic role of rare EpCAM-negative sphere forming cells.

Single-cell microRNA sequencing method comparison and application to cell lines and circulating lung tumor cells.

Molecular single cell analyses provide insights into physiological and pathological processes. Here, in a stepwise approach, we first evaluate 19 protocols for single cell small RNA sequencing on MCF7 cells spiked with 1 pg of 1,006 miRNAs. Second, we analyze MCF7 single cell equivalents of the eight best protocols. Third, we sequence single cells from eight different cell lines and 67 circulating tumor cells (CTCs) from seven SCLC patients. Altogether, we analyze 244 different samples. We observe high reproducibility within protocols and reads covered a broad spectrum of RNAs. For the 67 CTCs, we detect a median of 68 miRNAs, with 10 miRNAs being expressed in 90% of tested cells. Enrichment analysis suggested the lung as the most likely organ of origin and enrichment of cancer-related categories. Even the identification of non-annotated candidate miRNAs was feasible, underlining the potential of single cell small RNA sequencing.

Interleukin-6 trans-signaling is a candidate mechanism to drive progression of human DCCs during clinical latency.

Although thousands of breast cancer cells disseminate and home to bone marrow until primary surgery, usually less than a handful will succeed in establishing manifest metastases months to years later. To identify signals that support survival or outgrowth in patients, we profile rare bone marrow-derived disseminated cancer cells (DCCs) long before manifestation of metastasis and identify IL6/PI3K-signaling as candidate pathway for DCC activation. Surprisingly, and similar to mammary epithelial cells, DCCs lack membranous IL6 receptor expression and mechanistic dissection reveals IL6 trans-signaling to regulate a stem-like state of mammary epithelial cells via gp130. Responsiveness to IL6 trans-signals is found to be niche-dependent as bone marrow stromal and endosteal cells down-regulate gp130 in premalignant mammary epithelial cells as opposed to vascular niche cells. PIK3CA activation renders cells independent from IL6 trans-signaling. Consistent with a bottleneck function of microenvironmental DCC control, we find PIK3CA mutations highly associated with late-stage metastatic cells while being extremely rare in early DCCs. Our data suggest that the initial steps of metastasis formation are often not cancer cell-autonomous, but also depend on microenvironmental signals.

Microfluidic enrichment, isolation and characterization of disseminated melanoma cells from lymph node samples.

For the first time in melanoma, novel therapies have recently shown efficacy in the adjuvant therapy setting, which makes companion diagnostics to guide treatment decisions a desideratum. Early spread of disseminated cancer cells (DCC) to sentinel lymph nodes (SLN) is indicative of poor prognosis in melanoma and early DCCs could therefore provide important information about the malignant seed. Here, we present a strategy for enrichment of DCCs from SLN suspensions using a microfluidic device (Parsortix™, Angle plc). This approach enables the detection and isolation of viable DCCs, followed by molecular analysis and identification of genetic changes. By optimizing the workflow, the established protocol allows a high recovery of DCC from melanoma patient-derived lymph node (LN) suspensions with harvest rates above 60%. We then assessed the integrity of the transcriptome and genome of individual, isolated DCCs. In LNs of melanoma patients, we detected the expression of melanoma-associated transcripts including MLANA (encoding for MelanA protein), analyzed the BRAF and NRAS mutational status and confirmed the malignant origin of isolated melanoma DCCs by comparative genomic hybridization. We demonstrate the feasibility of epitope-independent isolation of LN DCCs using Parsortix™ for subsequent molecular characterization of isolated single DCCs with ample application fields including the use for companion diagnostics or subsequent cellular studies in personalized medicine.

Single cell polarity in liquid phase facilitates tumour metastasis.

Dynamic polarisation of tumour cells is essential for metastasis. While the role of polarisation during dedifferentiation and migration is well established, polarisation of metastasising tumour cells during phases of detachment has not been investigated. Here we identify and characterise a type of polarisation maintained by single cells in liquid phase termed single-cell (sc) polarity and investigate its role during metastasis. We demonstrate that sc polarity is an inherent feature of cells from different tumour entities that is observed in circulating tumour cells in patients. Functionally, we propose that the sc pole is directly involved in early attachment, thereby affecting adhesion, transmigration and metastasis. In vivo, the metastatic capacity of cell lines correlates with the extent of sc polarisation. By manipulating sc polarity regulators and by generic depolarisation, we show that sc polarity prior to migration affects transmigration and metastasis in vitro and in vivo.

Genetic alterations driving metastatic colony formation are acquired outside of the primary tumour in melanoma.

Mouse models indicate that metastatic dissemination occurs extremely early; however, the timing in human cancers is unknown. We therefore determined the time point of metastatic seeding relative to tumour thickness and genomic alterations in melanoma. Here, we find that lymphatic dissemination occurs shortly after dermal invasion of the primary lesion at a median thickness of ~0.5 mm and that typical driver changes, including BRAF mutation and gained or lost regions comprising genes like MET or CDKNA2, are acquired within the lymph node at the time of colony formation. These changes define a colonisation signature that was linked to xenograft formation in immunodeficient mice and death from melanoma. Thus, melanoma cells leave primary tumours early and evolve at different sites in parallel. We propose a model of metastatic melanoma dormancy, evolution and colonisation that will inform direct monitoring of adjuvant therapy targets.

The NAD+ precursor nicotinic acid improves genomic integrity in human peripheral blood mononuclear cells after X-irradiation.

NAD+ is an essential cofactor for enzymes catalyzing redox-reactions as well as an electron carrier in energy metabolism. Aside from this, NAD+ consuming enzymes like poly(ADP-ribose) polymerases and sirtuins are important regulators involved in chromatin-restructuring processes during repair and epigenetics/transcriptional adaption. In order to replenish cellular NAD+ levels after cleavage, synthesis starts from precursors such as nicotinamide, nicotinamide riboside or nicotinic acid to match the need for this essential molecule. In the present study, we investigated the impact of supplementation with nicotinic acid on resting and proliferating human mononuclear blood cells with a focus on DNA damage and repair processes. We observed that nicotinic acid supplementation increased NAD+ levels as well as DNA repair efficiency and enhanced genomic stability evaluated by micronucleus test after x-ray treatment. Interestingly, resting cells displayed lower basal levels of DNA breaks compared to proliferating cells, but break-induction rates were identical. Despite similar levels of p53 protein upregulation after irradiation, higher NAD+ concentrations led to reduced acetylation of this protein, suggesting enhanced SIRT1 activity. Our data reveal that even in normal primary human cells cellular NAD+ levels may be limiting under conditions of genotoxic stress and that boosting the NAD+ system with nicotinic acid can improve genomic stability.

DNA damage in Nijmegen Breakage Syndrome cells leads to PARP hyperactivation and increased oxidative stress.

Nijmegen Breakage Syndrome (NBS), an autosomal recessive genetic instability syndrome, is caused by hypomorphic mutation of the NBN gene, which codes for the protein nibrin. Nibrin is an integral member of the MRE11/RAD50/NBN (MRN) complex essential for processing DNA double-strand breaks. Cardinal features of NBS are immunodeficiency and an extremely high incidence of hematological malignancies. Recent studies in conditional null mutant mice have indicated disturbances in redox homeostasis due to impaired DSB processing. Clearly this could contribute to DNA damage, chromosomal instability, and cancer occurrence. Here we show, in the complete absence of nibrin in null mutant mouse cells, high levels of reactive oxygen species several hours after exposure to a mutagen. We show further that NBS patient cells, which unlike mouse null mutant cells have a truncated nibrin protein, also have high levels of reactive oxygen after DNA damage and that this increased oxidative stress is caused by depletion of NAD+ due to hyperactivation of the strand-break sensor, Poly(ADP-ribose) polymerase. Both hyperactivation of Poly(ADP-ribose) polymerase and increased ROS levels were reversed by use of a specific Poly(ADP-ribose) polymerase inhibitor. The extremely high incidence of malignancy among NBS patients is the result of the combination of a primary DSB repair deficiency with secondary oxidative DNA damage.

Ex vivo supplementation with nicotinic acid enhances cellular poly(ADP-ribosyl)ation and improves cell viability in human peripheral blood mononuclear cells.

Poly(ADP-ribosyl)ation is a posttranslational modification of proteins, which is mainly catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1) by using NAD(+) as substrate and is directly triggered by DNA strand breaks. Under mild genotoxic stress poly(ADP-ribose) (PAR) formation plays an important role in DNA repair whereas severe genotoxic stress and the ensuing overactivation of PARP-1 induce cellular NAD(+) depletion, energy failure and ultimately cell death. We are interested in studying the consequences of moderately enhanced enzymatic activity under conditions of DNA damage. Here we chose supplementation of cells with the NAD(+) precursor nicotinic acid (NA) as a strategy. In order to reliably assess PAR accumulation in living cells we first developed a novel, sensitive flow-cytometric method for the rapid analysis of poly(ADP-ribose) accumulation (RAPARA). Our data showed that ex vivo supplementation of human peripheral blood mononuclear cells (PBMC) with low concentrations of NA significantly raised cellular NAD(+) levels by 2.1-fold. Upon X-irradiation or exposure to hydrogen peroxide or N-methyl-N'-nitro-N-nitrosoguanidine, PAR accumulation was significantly increased and sustained in NA-supplemented cells. Furthermore, NA-supplemented PBMC displayed significantly higher cell viability due to a lower rate of necrotic cell death. In summary, ex vivo supplementation of human PBMC with NA increases cellular NAD(+) levels, boosts the cellular poly(ADP-ribosyl)ation response to genotoxic treatment, and protects from DNA-damage-induced cell death.