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.

A program of successive gene expression in mouse one-cell embryos.

At the moment of union in fertilization, sperm and oocyte are transcriptionally silent. The ensuing onset of embryonic transcription (embryonic genome activation [EGA]) is critical for development, yet its timing and profile remain elusive in any vertebrate species. We here dissect transcription during EGA by high-resolution single-cell RNA sequencing of precisely synchronized mouse one-cell embryos. This reveals a program of embryonic gene expression (immediate EGA [iEGA]) initiating within 4 h of fertilization. Expression during iEGA produces canonically spliced transcripts, occurs substantially from the maternal genome, and is mostly downregulated at the two-cell stage. Transcribed genes predict regulation by transcription factors (TFs) associated with cancer, including c-Myc. Blocking c-Myc or other predicted regulatory TF activities disrupts iEGA and induces acute developmental arrest. These findings illuminate intracellular mechanisms that regulate the onset of mammalian development and hold promise for the study of cancer.

Disseminated cancer cells detected by immunocytology in lymph nodes of NSCLC patients are highly prognostic and undergo parallel molecular evolution.

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 < 0.001). Assessment of all available 2,952 LNs of the same patients by HP uncovered additional patients escaping detection of lymphatic tumour spread by IC alone, consistent with the concept of skip metastasis. A combined lymph node status of IC and complete HP on a larger cohort of patients outperformed all risk factors in multivariable analysis for prognosis (p < 0.001; RR = 2.290; CI 1.407-3.728). Moreover, isolation of DCCs and single-cell molecular characterization revealed that (1) LN-DCCs differ from primary tumours in terms of copy number alterations and selected mutations and (2) critical alterations are acquired during colony formation within LNs. We conclude that LN-IC in NSCLC patients when combined with HP improves diagnostic precision, has the potential to reduce total workload, and facilitates molecular characterization of lymphatically spread cancer cells, which may become key for the selection and development of novel systemic therapies. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The clinical utility of cfRNA for disease detection and surveillance: A proof of concept study in non-small cell lung cancer.

BACKGROUND: CT scans are used in routine clinical practice for the diagnosis and treatment surveillance of non-small cell lung cancer (NSCLC). However, more sensitive methods are desirable. Liquid biopsy analyses of RNA and DNA can offer more sensitive diagnostic approaches. Cell-free RNA (cfRNA) has been described in several malignancies, but its clinical utility has not previously been explored. METHODS: We evaluated the clinical utility of cfRNA for early detection and surveillance of tumor disease in a proof-of-concept study. Using real-time-droplet digital polymerase chain reaction we characterized a candidate transcript (MORF4L2) in plasma samples from 41 advanced stage, 38 early stage NSCLC and 39 healthy samples. We compared its diagnostic performance with tumor markers and evaluated its utility for disease monitoring. RESULTS: MORF4L2 cfRNA was more abundant in patients than in healthy donors (p < 0.0001). Using the Youden index approach (cutoff value of 537 copies/ml was established) with a sensitivity of 0.73 (95% CI: 0.61-0.82) and a specificity of 0.87 (95% CI: 0.73-0.96). Positive and negative predictive values of 0.92 (95% CI: 0.83-0.95) and 0.59 (95% CI: 0.47-0.83) were achieved. Combination of cfRNA and Cyfra21-1 improved its predictive value from 89.5% to 94.7%. Low baseline MORF4L2 levels were associated with better overall survival (HR:0.25, 95% CI: 0.09-0.7, p = 0.009) and progression-free survival for patients treated with tyrosine kinase inhibitors (p = 0.011) and chemotherapy (p = 0.019). MORF4L2 profile between baseline and follow-up mirrored radiological response and tumor dynamics better than tumor markers. cfRNA transcripts allowed monitoring tumor dynamics in patients without tumor-reported genetic alterations. CONCLUSION: Our data support clinical utility of cfRNA for detection and surveillance of NSCLC. Further studies with larger cohorts are required.

EpCAM-positive disseminated cancer cells in bone marrow impact on survival of early-stage NSCLC patients.

INTRODUCTION: Detection of disseminated cancer cells (DCC) in bone marrow (BM) of patients with early-stage NSCLC has been associated with poor outcome. However, the phenotype, and hence relevant therapy targets, of DCCs in BM are unknown. We therefore compared a classical pan-Cytokeratin (CK) antibody for DCC detection with an anti-EpCAM antibody that may also detect more stem-like cells and tested whether assay positivity impacts on the survival of NSCLC patients. MATERIALS AND METHODS: We prospectively collected BM aspirates from 104 non-metastasized NSCLC patients that underwent potentially curative tumor resection from 2011 to 2016 at the Department of Thoracic Surgery of the University Hospital and Hospital Barmherzige Brüder in Regensburg. DCCs were detected by staining with the pan anti-CK antibody A45-B/B3 and the anti-EpCAM antibody HEA-125. We analyzed the association between detection of DCCs and clinicopathological characteristic and patient outcome. RESULTS: CK + and EpCAM + DCCs were detected in 45.2% and 52.9% of patients, respectively. Correlation between the two markers was low and neither of them was associated with sex, age, histology, T or N classification, resection status, grading or smoking habit. No significant association with tumor specific survival (TSS) and progression-free survival (PFS) was observed in patients with CK + DCCs. In contrast, detection of EpCAM + DCCs significantly correlated with reduced PFS (P = 0.017) and TSS (P = 0.017) and remained an independent prognostic variable for PFS and TSS upon multivariate testing (hazard ratio: 7.506 and 3.551, respectively). Detection of EpCAM + DCCs was the only prognostic marker for PFS. CONCLUSIONS: EpCAM+, but not CK + DCCs in BM predict reduced PFS and TSS. This finding suggests that EpCAM + DCCs in the BM comprise metastatic founder cells necessitating their in-depth molecular analysis for detection of novel therapy targets.

Combined multimodal ctDNA analysis and radiological imaging for tumor surveillance in Non-small cell lung cancer.

BACKGROUND: Radiology is the current standard for monitoring treatment responses in lung cancer. Limited sensitivity, exposure to ionizing radiations and related sequelae constitute some of its major limitation. Non-invasive and highly sensitive methods for early detection of treatment failures and resistance-associated disease progression would have additional clinical utility. METHODS: We analyzed serially collected plasma and paired tumor samples from lung cancer patients (61 with stage IV, 48 with stages I-III disease) and 61 healthy samples by means of next-generation sequencing, radiological imaging and droplet digital polymerase chain reaction (ddPCR) mutation and methylation assays. RESULTS: A 62% variant concordance between tumor-reported and circulating-free DNA (cfDNA) sequencing was observed between baseline liquid and tissue biopsies in stage IV patients. Interestingly, ctDNA sequencing allowed for the identification of resistance-mediating p.T790M mutations in baseline plasma samples for which no such mutation was observed in the corresponding tissue. Serial circulating tumor DNA (ctDNA) mutation analysis by means of ddPCR revealed a general decrease in ctDNA loads between baseline and first reassessment. Additionally, serial ctDNA analyses only recapitulated computed tomography (CT) -monitored tumor dynamics of some, but not all lesions within the same patient. To complement ctDNA variant analysis we devised a ctDNA methylation assay (methcfDNA) based on methylation-sensitive restriction enzymes. cfDNA methylation showed and area under the curve (AUC) of > 0.90 in early and late stage cases. A decrease in methcfDNA between baseline and first reassessment was reflected by a decrease in CT-derive tumor surface area, irrespective of tumor mutational status. CONCLUSION: Taken together, our data support the use of cfDNA sequencing for unbiased characterization of the molecular tumor architecture, highlights the impact of tumor architectural heterogeneity on ctDNA-based tumor surveillance and the added value of complementary approaches such as cfDNA methylation for early detection and monitoring.

Retrospective cell lineage reconstruction in humans by using short tandem repeats.

Cell lineage analysis aims to uncover the developmental history of an organism back to its cell of origin. Recently, novel in vivo methods utilizing genome editing enabled important insights into the cell lineages of animals. In contrast, human cell lineage remains restricted to retrospective approaches, which still lack resolution and cost-efficient solutions. Here, we demonstrate a scalable platform based on short tandem repeats targeted by duplex molecular inversion probes. With this human cell lineage tracing method, we accurately reproduced a known lineage of DU145 cells and reconstructed lineages of healthy and metastatic single cells from a melanoma patient who matched the anatomical reference while adding further refinements. This platform allowed us to faithfully recapitulate lineages of developmental tissue formation in healthy cells. In summary, our lineage discovery platform can profile informative somatic mutations efficiently and provides solid lineage reconstructions even in challenging low-mutation-rate healthy single 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.

Disseminated tumour cells from the bone marrow of early breast cancer patients: Results from an international pooled analysis.

PURPOSE: Presence of disseminated tumour cells (DTCs) in the bone marrow (BM) has been described as a surrogate of residual disease in patients with early breast cancer (EBC). PADDY (Pooled Analysis of DTC Detection in Early Breast Cancer) is a large international analysis of pooled data that aimed to assess the prognostic impact of DTCs in patients with EBC. EXPERIMENTAL DESIGN: Individual patient data were collected from 11 centres. Patients with EBC and available follow-up data in whom BM sampling was performed at the time of primary diagnosis before receiving any anticancer treatment were eligible. DTCs were identified by antibody staining against epithelial cytokeratins. Multivariate Cox regression was used to compare the survival of DTC-positive versus DTC-negative patients. RESULTS: In total, 10,307 patients were included. Of these, 2814 (27.3%) were DTC-positive. DTC detection was associated with higher tumour grade, larger tumour size, nodal positivity, oestrogen receptor and progesterone receptor negativity, and HER2 positivity (all p < 0.001). Multivariate analyses showed that DTC detection was an independent prognostic marker for overall survival, disease-free survival and distant disease-free survival with hazard ratios (HR) and 95% confidence intervals (CI) of 1.23 (95% CI: 1.06-1.43, p = 0.006), 1.30 (95% CI: 1.12-1.52, p < 0.001) and 1.30 (95% CI: 1.08-1.56, p = 0.006), respectively. There was no association between locoregional relapse-free survival and DTC detection (HR 1.21; 95% CI 0.68-2.16; p = 0.512). CONCLUSIONS: DTCs in the BM represent an independent prognostic marker in patients with EBC. The heterogeneous metastasis-initiating potential of DTCs is consistent with the concept of cancer dormancy.

Detection of Genomically Aberrant Cells within Circulating Tumor Microemboli (CTMs) Isolated from Early-Stage Breast Cancer Patients.

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.

Cancer-associated cells release citrate to support tumour metastatic progression.

Citrate is important for lipid synthesis and epigenetic regulation in addition to ATP production. We have previously reported that cancer cells import extracellular citrate via the pmCiC transporter to support their metabolism. Here, we show for the first time that citrate is supplied to cancer by cancer-associated stroma (CAS) and also that citrate synthesis and release is one of the latter's major metabolic tasks. Citrate release from CAS is controlled by cancer cells through cross-cellular communication. The availability of citrate from CAS regulated the cytokine profile, metabolism and features of cellular invasion. Moreover, citrate released by CAS is involved in inducing cancer progression especially enhancing invasiveness and organ colonisation. In line with the in vitro observations, we show that depriving cancer cells of citrate using gluconate, a specific inhibitor of pmCiC, significantly reduced the growth and metastatic spread of human pancreatic cancer cells in vivo and muted stromal activation and angiogenesis. We conclude that citrate is supplied to tumour cells by CAS and citrate uptake plays a significant role in cancer metastatic progression.

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.

Cancer progression and the invisible phase of metastatic colonization.

Metastatic dissemination occurs very early in the malignant progression of a cancer but the clinical manifestation of metastases often takes years. In recent decades, 5-year survival of patients with many solid cancers has increased due to earlier detection, local disease control and adjuvant therapies. As a consequence, we are confronted with an increase in late relapses as more antiproliferative cancer therapies prolong disease courses, raising questions about how cancer cells survive, evolve or stop growing and finally expand during periods of clinical latency. I argue here that the understanding of early metastasis formation, particularly of the currently invisible phase of metastatic colonization, will be essential for the next stage in adjuvant therapy development that reliably prevents metachronous metastasis.

Tumor Cell Invasion in Glioblastoma.

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.

Plasma Next Generation Sequencing and Droplet Digital-qPCR-Based Quantification of Circulating Cell-Free RNA for Noninvasive Early Detection of Cancer.

Early detection of cancer holds high promise for reducing cancer-related mortality. Detection of circulating tumor-specific nucleic acids holds promise, but sensitivity and specificity issues remain with current technology. We studied cell-free RNA (cfRNA) in patients with non-small cell lung cancer (NSCLC; n = 56 stage IV, n = 39 stages I-III), pancreatic cancer (PDAC, n = 20 stage III), malignant melanoma (MM, n = 12 stage III-IV), urothelial bladder cancer (UBC, n = 22 stage II and IV), and 65 healthy controls by means of next generation sequencing (NGS) and real-time droplet digital PCR (RT-ddPCR). We identified 192 overlapping upregulated transcripts in NSCLC and PDAC by NGS, more than 90% of which were noncoding. Previously reported transcripts (e.g., HOTAIRM1) were identified. Plasma cfRNA transcript levels of POU6F2-AS2 discriminated NSCLC from healthy donors (AUC = 0.82 and 0.76 for stages IV and I-III, respectively) and significantly associated (p = 0.017) with the established tumor marker Cyfra 21-1. cfRNA yield and POU6F2-AS transcript abundance discriminated PDAC patients from healthy donors (AUC = 1.0). POU6F2-AS2 transcript was significantly higher in MM (p = 0.044). In summary, our findings support further validation of cfRNA detection by RT-ddPCR as a biomarker for early detection of solid cancers.

Targeted transcript quantification in single disseminated cancer cells after whole transcriptome amplification.

Gene expression analysis of rare or heterogeneous cell populations such as disseminated cancer cells (DCCs) requires a sensitive method allowing reliable analysis of single cells. Therefore, we developed and explored the feasibility of a quantitative PCR (qPCR) assay to analyze single-cell cDNA pre-amplified using a previously established whole transcriptome amplification (WTA) protocol. We carefully selected and optimized multiple steps of the protocol, e.g. re-amplification of WTA products, quantification of amplified cDNA yields and final qPCR quantification, to identify the most reliable and accurate workflow for quantitation of gene expression of the ERBB2 gene in DCCs. We found that absolute quantification outperforms relative quantification. We then validated the performance of our method on single cells of established breast cancer cell lines displaying distinct levels of HER2 protein. The different protein levels were faithfully reflected by transcript expression across the tested cell lines thereby proving the accuracy of our approach. Finally, we applied our method to breast cancer DCCs of a patient undergoing anti-HER2-directed therapy. Here, we were able to measure ERBB2 expression levels in all HER2-protein-positive DCCs. In summary, we developed a reliable single-cell qPCR assay applicable to measure distinct levels of ERBB2 in DCCs.

Intravital imaging of glioma border morphology reveals distinctive cellular dynamics and contribution to tumor cell invasion.

The pathogenesis of glioblastoma (GBM) is characterized by highly invasive behavior allowing dissemination and progression. A conclusive image of the invasive process is not available. The aim of this work was to study invasion dynamics in GBM using an innovative in vivo imaging approach. Primary brain tumor initiating cell lines from IDH-wild type GBM stably expressing H2B-Dendra2 were implanted orthotopically in the brains of SCID mice. Using high-resolution time-lapse intravital imaging, tumor cell migration in the tumor core, border and invasive front was recorded. Tumor cell dynamics at different border configurations were analyzed and multivariate linear modelling of tumor cell spreading was performed. We found tumor border configurations, recapitulating human tumor border morphologies. Not only tumor borders but also the tumor core was composed of highly dynamic cells, with no clear correlation to the ability to spread into the brain. Two types of border configurations contributed to tumor cell spreading through distinct invasion patterns: an invasive margin that executes slow but directed invasion, and a diffuse infiltration margin with fast but less directed movement. By providing a more detailed view on glioma invasion patterns, our study may improve accuracy of prognosis and serve as a basis for personalized therapeutic approaches.

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.