Liquid Biopsy Detecting Circulating Tumor Cells in Patients with Non-Small Cell Lung Cancer: Preliminary Results of a Pilot Study.

Lung cancer is still the leading cause of cancer-related death worldwide. Interest is growing towards early detection and advances in liquid biopsy to isolate circulating tumor cells (CTCs). This pilot study aimed to detect epithelial CTCs in the peripheral blood of early-stage non-small cell lung cancer (NSCLC) patients. We used Smart BioSurface® (SBS) slide, a nanoparticle-coated slide able to immobilize viable nucleated cellular fraction without pre-selection and preserve cell integrity. Forty patients undergoing lung resection for NSCLC were included; they were divided into two groups according to CTC value, with a cut-off of three CTCs/mL. All patients were positive for CTCs. The mean CTC value was 4.7(± 5.8 S.D.) per ml/blood. In one patient, next generation sequencing (NGS) analysis of CTCs revealed v-raf murine sarcoma viral oncogene homolog B(BRAF) V600E mutation, which has also been identified in tissue biopsy. CTCs count affected neither overall survival (OS, p = 0.74) nor progression-free survival (p = 0.829). Multivariable analysis confirmed age (p = 0.020) and pNodal-stage (p = 0.028) as negative predictors of OS. Preliminary results of this pilot study suggest the capability of this method in detecting CTCs in all early-stage NSCLC patients. NGS on single cell, identified as CTC by immunofluorescence staining, is a powerful tool for investigating the molecular landscape of cancer, with the aim of personalized therapies.

Detection of clustered circulating tumour cells in early breast cancer.

Circulating tumour cell (CTC) clusters have been proposed to be major players in the metastatic spread of breast cancer, particularly during advanced disease stages. Yet, it is unclear whether or not they manifest in early breast cancer, as their occurrence in patients with metastasis-free primary disease has not been thoroughly evaluated. In this study, exploiting nanostructured titanium oxide-coated slides for shear-free CTC identification, we detect clustered CTCs in the curative setting of multiple patients with early breast cancer prior to surgical treatment, highlighting their presence already at early disease stages. These results spotlight an important aspect of metastasis biology and the possibility to intervene with anti-cluster therapeutics already during the early manifestation of breast cancer.

Antibody purification-independent microarrays (PIM) by direct bacteria spotting on TiO2-treated slides.

The preparation of effective conventional antibody microarrays depends on the availability of high quality material and on the correct accessibility of the antibody active moieties following their immobilization on the support slide. We show that spotting bacteria that expose recombinant antibodies on their external surface directly on nanostructured-TiO(2) or epoxy slides (purification-independent microarray - PIM) is a simple and reliable alternative for preparing sensitive and specific microarrays for antigen detection. Variable domains of single heavy-chain antibodies (VHHs) against fibroblast growth factor receptor 1 (FGFR1) were used to capture the antigen diluted in serum or BSA solution. The FGFR1 detection was performed by either direct antigen labeling or using a sandwich system in which FGFR1 was first bound to its antibody and successively identified using a labeled FGF. In both cases the signal distribution within each spot was uniform and spot morphology regular. The signal-to-noise ratio of the signal was extremely elevated and the specificity of the system was proved statistically. The LOD of the system for the antigen was calculated being 0.4ng/mL and the dynamic range between 0.4ng/mL and 10µg/mL. The microarrays prepared with bacteria exposing antibodies remain fully functional for at least 31 days after spotting. We finally demonstrated that the method is suitable for other antigen-antibody pairs and expect that it could be easily adapted to further applications such as the display of scFv and IgG antibodies or the autoantibody detection using protein PIMs.

Characterization of cluster-assembled nanostructured titanium oxide coatings as substrates for protein arrays.

Protein microarray technologies are rapidly expanding to fulfill current needs of proteome discovery for disease management. Nanostructured materials have been shown to present interesting features when used in biological settings: nanostructured titanium oxide film (ns-TiOx), synthesized by supersonic cluster beam deposition (SCBD), has recently emerged as a biocompatible substrate in different biological assays. The ns-TiOx surface is characterized by a morphology at the nanoscale that can be tuned to modulate specific biomolecule-material interactions. Here we present a systematic characterization of ns-TiOx coatings as protein binding surfaces, comparing their performances with those of most common commercial substrates in protein and antibody microarray assays. Through a robust statistical evaluation of repeatability in terms of coefficient of variation (CV) analysis, we demonstrate that ns-TiOx can be used as reliable substrate for biochips in analytical protein microarray application.