Purpose: The abundance and biological contribution of cancer-associated fibroblasts (CAF) in glioblastoma (GBM) are poorly understood. Here, we aim to uncover its molecular signature, cellular roles, and potential tumorigenesis implications. Experimental Design: We first applied single-cell RNA sequencing (RNA-seq) and bioinformatics analysis to identify and characterize stromal cells with CAF transcriptomic features in human GBM tumors. Then, we performed functional enrichment analysis and in vitro assays to investigate their interactions with malignant GBM cells. Results: We found that CAF abundance was low but significantly correlated with tumor grade, poor clinical outcome, and activation of extracellular matrix remodeling using three large cohorts containing bulk RNA-seq data and clinical information. Proteomic analysis of a GBM-derived CAF line and its secretome revealed fibronectin (FN1) as a critical candidate factor mediating CAF functions. This was validated using in vitro cellular models, which demonstrated that CAF-conditioned media and recombinant FN1 could facilitate the migration and invasion of GBM cells. In addition, we showed that CAFs were more abundant in the mesenchymal-like state (or subtype) than in other states of GBMs. Interestingly, cell lines resembling the proneural state responded to the CAF signaling better for the migratory and invasive phenotypes. Conclusions: Overall, this study characterized the molecular features and functional impacts of CAFs in GBM, alluding to novel cell interactions mediated by CAFs in the GBM microenvironment.

Numerous studies have shown that cell-free DNA (cfDNA) levels may serve as a non-invasive biomarker of a broad spectrum of acute and chronic pathologies. However, in order to make clinical decisions based on cfDNA measurements, it is essential to understand the magnitude of biological variation so this variation is not confused with a variation that actually represent a clinically relevant change. The present study was designed to evaluate the biological variation of cfDNA in healthy subjects and lung cancer patients. Plasma samples were collected from 33 healthy subjects and ten lung cancer patients over three days, as well as during the same day. CfDNA was quantified using droplet digital PCR. Biological variation data was estimated using mixed models. The within-subject variation was 25% and the between-subject variation was 30%. The reference change value for the healthy subjects was 70%. There was no systematic difference in cfDNA levels from day-to-day (p = 0⋅61), but there was a significant decline during the day (p0⋅3). Our findings show that cfDNA levels fluctuate significantly during the day and exhibit considerable within-subject variation. Thus, the data presented offer a substantial contribution to the interpretation of the clinical significance of cfDNA. Læge Sofus Carl Emil Friis og hustru Olga Doris Friis' Legat, Harboefonden, and Dagmar Marshalls Fond.

Objectives: The neurofilament light chain (NfL) has emerged as a versatile biomarker for CNS-diseases and is approaching clinical use. The observed changes in NfL levels are frequently of limited magnitude and in order to make clinical decisions based on NfL measurements, it is essential that biological variation is not confused with clinically relevant changes. The present study was designed to evaluate the biological variation of serum NfL. Methods: Apparently healthy individuals (n=33) were submitted to blood draws for three days in a row. On the second day, blood draws were performed every third hour for 12 h. NfL was quantified in serum using the Simoa™ HD-1 platform. The within-subject variation (CV_I) and between-subject variation (CV_G) were calculated using linear mixed-effects models. Results: The overall median value of NfL was 6.3 pg/mL (range 2.1–19.1). The CV_I was 3.1% and the CV_G was 35.6%. An increase in two serial measurements had to exceed 24.3% to be classified as significant at the 95% confidence level. Serum NfL levels remained stable during the day (p=0.40), whereas a minute variation (6.0–6.6 pg/mL) was observed from day-to-day (p=0.02). Conclusions: Serum NfL is subject to tight homeostatic regulation with none or neglectable semidiurnal and day-to-day variation, but considerable between-subject variation exists. This emphasizes serum NfL as a well-suited biomarker for disease monitoring, but warrants caution when interpreting NfL levels in relation to reference intervals in a diagnosis setting. Furthermore, NfL’s tight regulation requires that the analytical variation is kept at a minimum.

Although many epidermal growth factor receptor (EGFR)-mutated lung cancer patients initially benefit from the EGFR-inhibitor erlotinib, all acquire resistance. So far, several mechanisms implicated in resistance have been identified, but the existence of multiple resistance mechanisms in parallel have only been sparsely investigated. In this study, we investigated parallel resistance mechanisms acquired by HCC827, an EGFR-mutated adenocarcinoma cell line dependent on EGFR activity and sensitive to erlotinib. The cell line was treated with erlotinib by stepwise escalation of the drug-concentration and erlotinib-resistant (HCC827ER) cells created. HCC827ER cells depicted a mixed epithelial and mesenchymal phenotype. To clarify potential parallel resistance mechanisms, 14 resistant subclones were established by limited dilution. Interestingly, all HCC827ER subclones harbored either a MET-amplification (6/14) or underwent EMT (8/14), mechanisms both found in previous studies, but not in co-occurrence. Both subclone-types were resistant to erlotinib, but only MET-subclones responded to the MET-inhibitors crizotinib and capmatinib. EMT-subclones on the other hand had markedly increased FGFR1 expression and responded to the FGFR-inhibitor AZD4547, whereas MET-subclones did not. Monitoring gene expression through the development of HCC827ER revealed upregulation of FGFR1 expression as an early response to erlotinib. In addition, FGFR1 expression increased upon short-term erlotinib treatment (48 h) identifying a physiological role immediately after erlotinib exposure. The high FGFR1 expression seen in EMT-subclones was stable even after five passages without erlotinib. Here we show, that parallel resistance mechanisms appear during erlotinib-resistance development in EGFR-mutated NSCLC cells and highlight a role for FGFR1 expression changes as an early response to erlotinib as well as a bypass-signaling mechanism.

Background: Mutated circulating cell-free DNA (cfDNA) has been suggested as a surrogate marker of tumour burden and aggressiveness of disease. We examined the association between the level of plasma mutant cfDNA and metabolic tumour burden (MTB) measured by ¹⁸F-fluoro-D-glucose positron emission tomography/computed tomography (¹⁸F-FDG PET/CT). Furthermore, the presence of mutant cfDNA was correlated with patient survival. Methods: Forty-six advanced non-small cell lung cancer (NSCLC) patients were included. At the time of inclusion, blood sampling and a PET/CT scan were performed. cfDNA was isolated and next-generation sequencing (NGS) was performed (Ion AmpliSeq Colon and Lung Cancer panel v2). MTB was defined by a volumetric PET parameter. Results: NGS succeeded in 41 patients. Mutations were detected in the blood of 24 patients. A significant correlation between the allele frequency of the most frequent mutation and MTB was found (P=0.001). Patients with detectable mutated cfDNA had a significantly shorter median overall survival compared with patients without (3.7 versus 10.6 months, P=0.019). This impact on survival was independent of the MTB. Conclusions: Level of mutated cfDNA tends to correlate with MTB in advanced-stage NSCLC patients. Patients with detectable mutant DNA in plasma had an inferior survival, indicating that this could be an important predictor of survival.