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In melanoma, immunocytology (IC) after sentinel lymph node disaggregation not only enables better quantification of disseminated cancer cells (DCCs) than routine histopathology (HP) but also provides a unique opportunity to detect, isolate, and analyse these earliest harbingers of metachronous metastasis. Here, we explored lymph node IC in non‐small cell lung cancer (NSCLC). For 122 NSCLC patients, 220 lymph nodes (LNs) were split in half and prepared for IC and HP. When both methods were compared, IC identified 22% positive patients as opposed to 4.5% by HP, revealing a much higher sensitivity of IC (p

Simple Summary Distant metastases derive from the shedding and dissemination of single cancer cells (CTCs) or circulating tumor emboli (CTMs) into circulation. Previous studies on CTMs were mainly run in patients with metastatic disease; however, we observed that CTMs are more frequently detected in patients with early-stage breast cancer. Here, we collected single CTMs and their relative primary tumor tissue samples in early-stage patients. By studying genomic aberrations, present in tumors cells and absent in normal cells, we predicted the tumor fraction thanks to a statistical model developed from a calibration curve with breast cancer cell lines. The tumor fraction ranged from 8% to 48% and CTMs contained specific and shared alterations with respect to tissue. Thus, CTMs may derive from different regions of the primary tumor or from occult micrometastases. Moreover, CTM-private mutations may inform us about specific metastasis-associated functions of involved genes that should be further explored in follow-up and mechanistic studies. Abstract Circulating tumor microemboli (CTMs) are clusters of cancer cells detached from solid tumors, whose study can reveal mechanisms underlying metastatization. As they frequently comprise unknown fractions of leukocytes, the analysis of copy number alterations (CNAs) is challenging. To address this, we titrated known numbers of leukocytes into cancer cells (MDA-MB-453 and MDA-MB-36, displaying high and low DNA content, respectively) generating tumor fractions from 0–100%. After low-pass sequencing, ichorCNA was identified as the best algorithm to build a linear mixed regression model for tumor fraction (TF) prediction. We then isolated 53 CTMs from blood samples of six early-stage breast cancer patients and predicted the TF of all clusters. We found that all clusters harbor cancer cells between 8 and 48%. Furthermore, by comparing the identified CNAs of CTMs with their matched primary tumors, we noted that only 31–71% of aberrations were shared. Surprisingly, CTM-private alterations were abundant (30–63%), whereas primary tumor-private alterations were rare (4–12%). This either indicates that CTMs are disseminated from further progressed regions of the primary tumor or stem from cancer cells already colonizing distant sites. In both cases, CTM-private mutations may inform us about specific metastasis-associated functions of involved genes that should be explored in follow-up and mechanistic studies.

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., Metastatic dissemination in breast cancer patients occurs early in malignant transformation, raising questions about how disseminated cancer cells (DCC) progress at distant sites. Here, the authors show that DCCs in bone marrow are activated via IL6-trans-signaling and thereby acquire stemness traits relevant for metastasis formation.

Glioblastoma (GBM) is a particularly devastating tumor with a median survival of about 16 months. Recent research has revealed novel insights into the outstanding heterogeneity of this type of brain cancer. However, all GBM subtypes share the hallmark feature of aggressive invasion into the surrounding tissue. Invasive glioblastoma cells escape surgery and focal therapies and thus represent a major obstacle for curative therapy. This review aims to provide a comprehensive understanding of glioma invasion mechanisms with respect to tumor-cell-intrinsic properties as well as cues provided by the microenvironment. We discuss genetic programs that may influence the dissemination and plasticity of GBM cells as well as their different invasion patterns. We also review how tumor cells shape their microenvironment and how, vice versa, components of the extracellular matrix and factors from non-neoplastic cells influence tumor cell motility. We further discuss different research platforms for modeling invasion. Finally, we highlight the importance of accounting for the complex interplay between tumor cell invasion and treatment resistance in glioblastoma when considering new therapeutic approaches.

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.

Background: Clinical management of cancers of the central nevus system (CNS) is very challenging as they often exhibit low responsiveness to radiation and chemotherapy resulting in overall poor survival. Moreover, analysis of mechanisms driving cancer progression and selection of targeted therapies in CNS tumors is hindered by the limited availability to tumor tissues accessible only though surgical biopsies. A potential source of cancer material in patients with CNS is cerebrospinal fluid (CSF). Analysis of CSF-derived disseminated cancer cells (csfDCCs) holds a promise for improvement of diagnostics and monitoring of CNS tumors. For this reason, we developed a novel workflow allowing detection, isolation and multi-omic analysis of csfDCCs. Methods: In a proof of concept study a new workflow was used to analyze CSF samples from two patients with medulloblastoma and pineoblastoma. CSF-derived cells were stained for CD45 to allow identification of infiltrating immune cells. Putative csfDCCs (CD45-negative) and control cells (CD45-positive) were subjected to a multi-omic workflow allowing parallel sequencing of genomes and transcriptomes of the same cells. Single-cell mRNA was physically separated from DNA, amplified by means of whole transcriptome amplification (Ampli1 WTA) and analyzed using endpoint PCR and a proprietary single-cell RNA-Seq approach. In parallel, DNA was subjected to whole genome amplification (Ampli1 WGA) and analyzed for the presence of copy number variations as well as point mutations (Ampli1 LowPass Kit and targeted sequencing of actionable hot-spots). Results: We analyzed seven CNS-derived single cells and five cell clusters from the medulloblastoma patient and further eight cell clusters and eight single cells obtained from the pineoblastoma patient. Transcriptome analysis revealed that expression of neural lineage markers (e.g. CD133, SYP, OTX2, MSI1, MAP2, NEUROG1 and NEUROD1) is present almost exclusively in CD45-negative cells. Only two samples collected from medulloblastoma patient co-expressed CD45 and GFAP. However, DNA analysis revealed that CD45-positive and CD45/GFAP double-positive samples showed non-aberrant genomic profiles, thus these cells were classified as non-malignant. In contrast, all CD45-negative cells harbored genetic alterations confirming their malignant origin. Conclusion: Our proof of concept study shows a novel workflow allowing identification, isolation as well as parallel genome and transcriptome analysis csfDCCs of patients with pediatric CNS tumors. Citation Format: Zbigniew T. Czyz, Giancarlo Feliciello, Miodrag Guzvic, Thomas Schamberger, Sandra Grunewald, Selim Corbacioglu, Markus J. Riemenschneider, Bernhard Polzer, Christoph A. Klein. A novel workflow for isolation and multi-omic profiling of DCCs derived from cerebrospinal fluid of patients with pediatric brain cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-239.

We recently showed that lymph node disaggregation followed by immunocytology enables precise quantification of disseminated cancer cells (DCCs) and demonstrated that this approach has a 20-fold higher sensitivity to detect melanoma DCCs than routine histopathology (Ulmer et al., PLoS Medicine, 2014). Moreover, genetic profiling of single melanoma DCCs identified a colonization signature consisting of specific copy number alterations and point mutations that identify patients with high risk of progression (Werner-Klein et al., Nature Communications 2018). Here, we present the adaptation of this method to a semi-automated workflow for detection, isolation and molecular analysis of single melanoma DCCs. The developed workflow includes a mechanical disaggregation of lymph node tissue and collection of the mononuclear cells, immunofluorescence staining against melanoma-associated markers gp100 and MCSP and depletion of CD45-positive cells. Individual melanoma cells are then detected and isolated by DEPArrayTM technology enabling single cell whole genome amplification (Ampli1TM) for subsequent molecular analysis. In total, we processed 20 lymph nodes of melanoma patients and detected melanoma DCCs in 11/20 samples (55%). The quality of isolated cells was checked by Ampli1TM QC and 174 isolated single cells were further analyzed by Sanger sequencing for specific point mutations (BRAF, NRAS and cKIT) and Ampli1TM low pass kit for Illumina for copy number variation (CNV). Successful molecular analysis was correlated with genome integrity score (GII) as determined by Ampli1TM QC, with more than 95% of cells with GII 3-4 showing good performance in low pass sequencing. In conclusion, a new DepArrayTM based application for marker-dependent single cell isolation from malignant melanoma lymph nodes was successfully established and tested on a cohort of 20 melanoma patients. Molecular analyses of isolated single cells confirmed the tumor origin by CNV profiling and mutational analysis of melanoma-associated mutations. In the future, this approach could help to select individualized therapies for melanoma patients. Citation Format: Barbara Alberter, Sebastian Scheitler, Giancarlo Feliciello, Alberto Ferrarini, Melanie Werner-Klein, Sebastian Haferkamp, Christoph A. Klein, Bernhard Polzer. Semi-automated detection, isolation and molecular analysis of single disseminated melanoma cells from lymph nodes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 431.

Introduction: Manifestation of bone metastases is the most frequent cause of death among prostate cancer patients. Since the progression from non-metastatic (M0) to metastatic (M1) is highly unpredictable, molecular analysis of disseminated cancer cells (DCCs) detected before overt metastasis may provide an opportunity to dissect the early stages of systemic cancer and enable detection of critical therapy targets. However, we previously showed that early stage prostate DCCs acquire a bone marrow-associated transcriptomic phenotype (Guzvic et al. Cancer Res. 2014, 15;74(24):7383-94) impeding the definite identification of DCCs. To address this problem we developed a workflow of combined single-cell RNA-Seq and single-cell aCGH allowing high-resolution analysis of genomes and transcriptomes of individual cells. Here, we present a proof-of-concept study using DCCs isolated from bone marrow of prostate cancer patients. Materials and methods: We isolated 24 EPCAM-positive DCCs from bone marrow of prostate cancer patients. In addition, we analyzed two VCaP prostate cancer cells and two peripheral blood lymphocytes of healthy individuals. All isolated cells were subjected to combined whole genome and whole transcriptome amplification using Ampli1TM WGA Kit and a WTA approach (Klein CA et al., Nat Biotechnol. 2002, 20(4):387-92), respectively. The suitability of WTA and WGA products for downstream analyses were assessed using PCR-based QC-assays. Subsequently, WGA products were hybridized on high-resolution SurePrint aCGH arrays and analyzed with Agilent Genomic Workbench Software. WTA products were sequenced using Roche 454 GS FLX+ or Illumina HiSeq 2000 systems. Single-cell RNA-Seq data were evaluated using a bioinformatic pipeline adjusted to the needs of the single cell WTA method. Results: We provide two experimental protocols for single-cell RNA-Seq: (i) allowing detection of low-abundant transcripts and (ii) enabling comprehensive gene-expression analysis. The protocols were validated using VCaP cells, in which we could successfully detect low expression levels of TMPRSS2-ERG fusion transcripts. The established analysis allowed also detection of novel fusion transcripts in a M1-stage prostate cancer patient. Comprehensive gene expression analysis revealed presence of 4.000 to 13.000 transcripts expressed in bone marrow-derived cells. The comparison of RNA-Seq and aCGH data allowed to simultaneously detecting copy number alterations and corresponding changes in gene expression dosage. Thereby, the combined genome and transcriptome analysis of single cells enables to uncover the impact of genomic gains and losses on cellular transcriptomes. Conclusions: Combined genome and transcriptome analysis of patient-derived single DCCs is feasible and enables unambiguous identification and genotypic and phenotypic characterization of DCCs. Citation Format: Stefan Kirsch, Urs Lahrmann, Miodrag Guzvic, Zbigniew T. Czyz, Giancarlo Feliciello, Bernhard Polzer, Christoph A. Klein. High-resolution parallel analysis of genome and transcriptome of single disseminated prostate cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1551.