Mutation analysis in individual circulating tumor cells depicts intratumor heterogeneity in melanoma.

Circulating tumor DNA (ctDNA) is the cornerstone of liquid biopsy diagnostics, revealing clinically relevant genomic aberrations from blood of cancer patients. Genomic analysis of single circulating tumor cells (CTCs) could provide additional insights into intra-patient heterogeneity, but it requires whole-genome amplification (WGA) of DNA, which might introduce bias. Here, we describe a novel approach based on mass spectrometry for mutation detection from individual CTCs not requiring WGA and complex bioinformatics pipelines. After establishment of our protocol on tumor cell line-derived single cells, it was validated on CTCs of 33 metastatic melanoma patients and the mutations were compared to those obtained from tumor tissue and ctDNA. Although concordance with tumor tissue was superior for ctDNA over CTC analysis, a larger number of mutations were found within CTCs compared to ctDNA (p = 0.039), including mutations in melanoma driver genes, or those associated with resistance to therapy or metastasis. Thus, our results demonstrate proof-of-principle data that CTC analysis can provide clinically relevant genomic information that is not redundant to tumor tissue or ctDNA analysis.

Heparan sulfate dependent binding of plasmatic von Willebrand factor to blood circulating melanoma cells attenuates metastasis.

Heparan sulfate (HS), a highly negatively charged glycosaminoglycan, is ubiquitously present in all tissues and also exposed on the surface of mammalian cells. A plethora of molecules such as growth factors, cytokines or coagulation factors bear HS binding sites. Accordingly, HS controls the communication of cells with their environment and therefore numerous physiological and pathophysiological processes such as cell adhesion, migration, and cancer cell metastasis. In the present work, we found that HS exposed by blood circulating melanoma cells recruited considerable amounts of plasmatic von Willebrand factor (vWF) to the cellular surface. Analyses assisted by super-resolution microscopy indicated that HS and vWF formed a tight molecular complex. Enzymatic removal of HS or genetic engineering of the HS biosynthesis showed that a reduced length of the HS chains or complete lack of HS was associated with significantly reduced vWF encapsulation. In microfluidic experiments, mimicking a tumor-activated vascular system, we found that vWF-HS complexes prevented vascular adhesion. In line with this, single molecular force spectroscopy suggested that the vWF-HS complex promoted the repulsion of circulating cancer cells from the blood vessel wall to counteract metastasis. Experiments in wild type and vWF knockout mice confirmed that the HS-vWF complex at the melanoma cell surface attenuated hematogenous metastasis, whereas melanoma cells lacking HS evade the anti-metastatic recognition by vWF. Analysis of tissue samples obtained from melanoma patients validated that metastatic melanoma cells produce less HS. Transcriptome data further suggest that attenuated expression of HS-related genes correlate with metastases and reduced patients' survival. In conclusion, we showed that HS-mediated binding of plasmatic vWF to the cellular surface can reduce the hematogenous spread of melanoma. Cancer cells with low HS levels evade vWF recognition and are thus prone to form metastases. Therefore, therapeutic expansion of the cancer cell exposed HS may prevent tumor progression.

Increased replication stress and R-loop accumulation in EGFRvIII-expressing glioblastoma present new therapeutic opportunities.

Background: The oncogene epidermal growth factor receptor variant III (EGFRvIII) is expressed in approximately one-third of all glioblastomas (GBMs). So far it is not clear if EGFRvIII expression induces replication stress in GBM cells, which might serve as a therapeutical target. Methods: Isogenetic EGFRvIII- and EGFRvIII+ cell lines with endogenous EGFRvIII expression were used. Markers of oncogenic and replication stress such as γH2AX, RPA, 53BP1, ATR, and CHK1 were analyzed using western blot, immunofluorescence, and flow cytometry. The DNA fiber assay was performed to analyze replication, transcription was measured by incorporation of EU, and genomic instability was investigated by micronuclei and CGH-Array analysis. Immunohistochemistry staining was used to detect replication stress markers and R-loops in human GBM samples. Results: EGFRvIII+ cells exhibit an activated replication stress response, increased spontaneous DNA damage, elevated levels of single-stranded DNA, and reduced DNA replication velocity, which are all indicative characteristics of replication stress. Furthermore, we show here that EGFRvIII expression is linked to increased genomic instability. EGFRvIII-expressing cells display elevated RNA synthesis and R-loop formation, which could also be confirmed in EGFRvIII-positive GBM patient samples. Targeting replication stress by irinotecan resulted in increased sensitivity of EGFRvIII+ cells. Conclusion: This study demonstrates that EGFRvIII expression is associated with increased replication stress, R-loop accumulation, and genomic instability. This might contribute to intratumoral heterogeneity but may also be exploited for individualized therapy approaches.

MicroRNAs from Liquid Biopsy Derived Extracellular Vesicles: Recent Advances in Detection and Characterization Methods.

Liquid biopsies have become a convenient tool in cancer diagnostics, real-time disease monitoring, and evaluation of residual disease. Yet, the information still encrypted in the variety of tumor-derived molecules identified in biofluids has proven difficult to decipher due to the technological limitations imposed by their biological nature. Such is the case of extracellular vesicle (EV) encapsulated ncRNAs, which have gained traction in recent years as biomarkers. Due to their resilience towards degrading factors they may act as suitable disease indicators. This review addresses the less described issues in this context. We present an overview of less investigated biofluids that can be used for EV isolation in addition to different isolation approaches to overcome the technical challenges these specimens harbor. Furthermore, we summarize the latest technological advances providing improvement to ncRNA detection and analysis. Thereby, this review summarizes the current state-of-the-art methodologies regarding EV and EV derived miRNA analysis and how they compare to current approaches.

Circulating Non-coding RNAs in Renal Cell Carcinoma-Pathogenesis and Potential Implications as Clinical Biomarkers.

Liquid biopsy-the determination of circulating cells, proteins, DNA or RNA from biofluids through a "less invasive" approach-has emerged as a novel approach in all cancer entities. Circulating non-(protein) coding RNAs including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and YRNAs can be passively released by tissue or cell damage or actively secreted as cell-free circulating RNAs, bound to lipoproteins or carried by exosomes. In renal cell carcinoma (RCC), a growing body of evidence suggests circulating non-coding RNAs (ncRNAs) such as miRNAs, lncRNAs, and YRNAs as promising and easily accessible blood-based biomarkers for the early diagnosis of RCC as well as for the prediction of prognosis and treatment response. In addition, circulating ncRNAs could also play a role in RCC pathogenesis and progression. This review gives an overview over the current study landscape of circulating ncRNAs and their involvement in RCC pathogenesis as well as their potential utility as future biomarkers in RCC diagnosis and treatment.

Evaluation of PD-L1 expression on circulating tumor cells (CTCs) in patients with advanced urothelial carcinoma (UC).

Immune checkpoint inhibition (ICI) of the PD-1/PD-L1 axis shows durable responses in a subset of patients with metastatic urothelial carcinoma (UC). However, PD-L1 expression in tumor biopsies does not necessarily correlate with response to PD-1/PD-L1 inhibitors. Thus, a reliable predictive biomarker is urgently needed. Here, the expression of PD-L1 on circulating tumor cells (CTCs) in blood from patients with advanced UC was analyzed. For this purpose, an assay to test PD-L1 expression on CTCs using the CellSearch® system was established using cells of five UC cell lines spiked into blood samples from healthy donors and applied to a heterogeneous cohort of UC patients. Enumeration of CTCs was performed in blood samples from 49 patients with advanced UC. PD-L1 expression in ≥1 CTC was found in 10 of 16 CTC-positive samples (63%). Both intra- and inter-patient heterogeneity regarding PD-L1 expression of CTCs were observed. Furthermore, vimentin-expressing CTCs were detected in 4 of 15 CTC-positive samples (27%), independently of PD-L1 analysis. Both CTC detection and presence of CTCs with moderate or strong PD-L1 expression correlated with worse overall survival. Analyses during disease course of three individual patients receiving ICI suggest that apart from CTC numbers also PD-L1 expression on CTCs might potentially indicate disease progression. This is the first study demonstrating the feasibility to detect CTC-PD-L1 expression in patients with advanced UC using the CellSearch® system. This assay is readily available for clinical application and could be implemented in future clinical trials to evaluate its relevance for predicting and monitoring response to ICI.

FuncPEP: A Database of Functional Peptides Encoded by Non-Coding RNAs.

Non-coding RNAs (ncRNAs) are essential players in many cellular processes, from normal development to oncogenic transformation. Initially, ncRNAs were defined as transcripts that lacked an open reading frame (ORF). However, multiple lines of evidence suggest that certain ncRNAs encode small peptides of less than 100 amino acids. The sequences encoding these peptides are known as small open reading frames (smORFs), many initiating with the traditional AUG start codon but terminating with atypical stop codons, suggesting a different biogenesis. The ncRNA-encoded peptides (ncPEPs) are gradually becoming appreciated as a new class of functional molecules that contribute to diverse cellular processes, and are deregulated in different diseases contributing to pathogenesis. As multiple publications have identified unique ncPEPs, we appreciated the need for assembling a new web resource that could gather information about these functional ncPEPs. We developed FuncPEP, a new database of functional ncRNA encoded peptides, containing all experimentally validated and functionally characterized ncPEPs. Currently, FuncPEP includes a comprehensive annotation of 112 functional ncPEPs and specific details regarding the ncRNA transcripts that encode these peptides. We believe that FuncPEP will serve as a platform for further deciphering the biologic significance and medical use of ncPEPs.

EGFRvIII upregulates DNA mismatch repair resulting in increased temozolomide sensitivity of MGMT promoter methylated glioblastoma.

The oncogene epidermal growth factor receptor variant III (EGFRvIII) is frequently expressed in glioblastomas (GBM) but its impact on therapy response is still under controversial debate. Here we wanted to test if EGFRvIII influences the sensitivity towards the alkylating agent temozolomide (TMZ). Therefore, we retrospectively analyzed the survival of 336 GBM patients, demonstrating that under standard treatment, which includes TMZ, EGFRvIII expression is associated with prolonged survival, but only in patients with O6-methylguanine-DNA methyltransferase (MGMT) promoter methylated tumors. Using isogenic GBM cell lines with endogenous EGFRvIII expression we could demonstrate that EGFRvIII increases TMZ sensitivity and results in enhanced numbers of DNA double-strand breaks and a pronounced S/G2-phase arrest after TMZ treatment. We observed a higher expression of DNA mismatch repair (MMR) proteins in EGFRvIII+ cells and patient tumor samples, which was most pronounced for MSH2 and MSH6. EGFRvIII-specific knockdown reduced MMR protein expression thereby increasing TMZ resistance. Subsequent functional kinome profiling revealed an increased activation of p38- and ERK1/2-dependent signaling in EGFRvIII expressing cells, which regulates MMR protein expression downstream of EGFRvIII. In summary, our results demonstrate that the oncoprotein EGFRvIII sensitizes a fraction of GBM to current standard of care treatment through the upregulation of DNA MMR.

Characterization of circulating breast cancer cells with tumorigenic and metastatic capacity.

Functional studies giving insight into the biology of circulating tumor cells (CTCs) remain scarce due to the low frequency of CTCs and lack of appropriate models. Here, we describe the characterization of a novel CTC-derived breast cancer cell line, designated CTC-ITB-01, established from a patient with metastatic estrogen receptor-positive (ER+ ) breast cancer, resistant to endocrine therapy. CTC-ITB-01 remained ER+ in culture, and copy number alteration (CNA) profiling showed high concordance between CTC-ITB-01 and CTCs originally present in the patient with cancer at the time point of blood draw. RNA-sequencing data indicate that CTC-ITB-01 has a predominantly epithelial expression signature. Primary tumor and metastasis formation in an intraductal PDX mouse model mirrored the clinical progression of ER+ breast cancer. Downstream ER signaling was constitutively active in CTC-ITB-01 independent of ligand availability, and the CDK4/6 inhibitor Palbociclib strongly inhibited CTC-ITB-01 growth. Thus, we established a functional model that opens a new avenue to study CTC biology.