Non-Small Cell Lung Cancer Testing on Reference Specimens: An Italian Multicenter Experience.

INTRODUCTION: Biomarker testing is mandatory for the clinical management of patients with advanced non-small cell lung cancer (NSCLC). Myriads of technical platforms are now available for biomarker analysis with differences in terms of multiplexing capability, analytical sensitivity, and turnaround time (TAT). We evaluated the technical performance of the diagnostic workflows of 24 representative Italian institutions performing molecular tests on a series of artificial reference specimens built to mimic routine diagnostic samples. METHODS: Sample sets of eight slides from cell blocks of artificial reference specimens harboring exon 19 EGFR (epidermal growth factor receptor) p.E746_AT50del, exon 2 KRAS (Kirsten rat sarcoma viral oncogene homologue) p.G12C, ROS1 (c-ros oncogene 1)-unknown gene fusion, and MET (MET proto-oncogene, receptor tyrosine kinase) Δ exon 14 skipping were distributed to each participating institution. Two independent cell block specimens were validated by the University of Naples Federico II before shipment. Methodological and molecular data from reference specimens were annotated. RESULTS: Overall, a median DNA concentration of 3.3 ng/µL (range 0.1-10.0 ng/µL) and 13.4 ng/µL (range 2.0-45.8 ng/µL) were obtained with automated and manual technical procedures, respectively. RNA concentrations of 5.7 ng/µL (range 0.2-11.9 ng/µL) and 9.3 ng/µL (range 0.5-18.0 ng/µL) were also detected. KRAS exon 2 p.G12C, EGFR exon 19 p.E736_A750del hotspot mutations, and ROS1 aberrant transcripts were identified in all tested cases, whereas 15 out of 16 (93.7%) centers detected MET exon 14 skipping mutation. CONCLUSIONS: Optimized technical workflows are crucial in the decision-making strategy of patients with NSCLC. Artificial reference specimens enable optimization of diagnostic workflows for predictive molecular analysis in routine clinical practice.

Celecoxib, a Non-Steroidal Anti-Inflammatory Drug, Exerts a Toxic Effect on Human Melanoma Cells Grown as 2D and 3D Cell Cultures.

Cutaneous melanoma (CM) remains one of the leading causes of tumor mortality due to its high metastatic spread. CM growth is influenced by inflammation regulated by prostaglandins (PGs) whose synthesis is catalyzed by cyclooxygenases (COXs). COX inhibitors, including non-steroidal anti-inflammatory drugs (NSAIDs), can inhibit tumor development and growth. In particular, in vitro experiments have shown that celecoxib, a NSAID, inhibits the growth of some tumor cell lines. However, two-dimensional (2D) cell cultures, used in traditional in vitro anticancer assays, often show poor efficacy due to a lack of an in vivo like cellular environment. Three-dimensional (3D) cell cultures, such as spheroids, are better models because they can mimic the common features displayed by human solid tumors. Hence, in this study, we evaluated the anti-neoplastic potential of celecoxib, in both 2D and 3D cell cultures of A2058 and SAN melanoma cell lines. In particular, celecoxib reduced the cell viability and migratory capability and triggered the apoptosis of melanoma cells grown as 2D cultures. When celecoxib was tested on 3D melanoma cell cultures, the drug exerted an inhibitory effect on cell outgrowth from spheroids and reduced the invasiveness of melanoma cell spheroids into the hydrogel matrix. This work suggests that celecoxib could represent a new potential therapeutic approach in melanoma therapy.

Thyroid Cancer and Fibroblasts.

Thyroid cancer is the most common type of endocrine cancer, and its prevalence continue to rise. Non-metastatic thyroid cancer patients are successfully treated. However, looking for new therapeutic strategies is of great importance for metastatic thyroid cancers that still lead to death. With respect to this, the tumor microenvironment (TME), which plays a key role in tumor progression, should be considered as a new promising therapeutic target to hamper thyroid cancer progression. Indeed, thyroid tumors consist of cancer cells and a heterogeneous and ever-changing niche, represented by the TME, which contributes to establishing most of the features of cancer cells. The TME consists of extracellular matrix (ECM) molecules, soluble factors, metabolites, blood and lymphatic tumor vessels and several stromal cell types that, by interacting with each other and with tumor cells, affect TME remodeling, cancer growth and progression. Among the thyroid TME components, cancer-associated fibroblasts (CAFs) have gained more attention in the last years. Indeed, recent important evidence showed that thyroid CAFs strongly sustain thyroid cancer growth and progression by producing soluble factors and ECM proteins, which, in turn, deeply affect thyroid cancer cell behavior and aggressiveness. Hence, in this article, we describe the thyroid TME, focusing on the desmoplastic stromal reaction, which is a powerful indicator of thyroid cancer progression and an invasive growth pattern. In addition, we discuss the origins and features of the thyroid CAFs, their influence on thyroid cancer growth and progression, their role in remodeling the ECM and their immune-modulating functions. We finally debate therapeutic perspectives targeting CAFs.

Generation and Characterization of a Tumor Stromal Microenvironment and Analysis of Its Interplay with Breast Cancer Cells: An In Vitro Model to Study Breast Cancer-Associated Fibroblast Inactivation.

Breast cancer-associated fibroblasts (BCAFs), the most abundant non-cancer stromal cells of the breast tumor microenvironment (TME), dramatically sustain breast cancer (BC) progression by interacting with BC cells. BCAFs, as well as myofibroblasts, display an up regulation of activation and inflammation markers represented by α-smooth muscle actin (α-SMA) and cyclooxygenase 2 (COX-2). BCAF aggregates have been identified in the peripheral blood of metastatic BC patients. We generated an in vitro stromal model consisting of human primary BCAFs grown as monolayers or 3D cell aggregates, namely spheroids and reverted BCAFs, obtained from BCAF spheroids reverted to 2D cell adhesion growth after 216 h of 3D culture. We firstly evaluated the state of activation and inflammation and the mesenchymal status of the BCAF monolayers, BCAF spheroids and reverted BCAFs. Then, we analyzed the MCF-7 cell viability and migration following treatment with conditioned media from the different BCAF cultures. After 216 h of 3D culture, the BCAFs acquired an inactivated phenotype, associated with a significant reduction in α-SMA and COX-2 protein expression. The deactivation of the BCAF spheroids at 216 h was further confirmed by the cytostatic effect exerted by their conditioned medium on MCF-7 cells. Interestingly, the reverted BCAFs also retained a less activated phenotype as indicated by α-SMA protein expression reduction. Furthermore, the reverted BCAFs exhibited a reduced pro-tumor phenotype as indicated by the anti-migratory effect exerted by their conditioned medium on MCF-7 cells. The deactivation of BCAFs without drug treatment is possible and leads to a reduced capability of BCAFs to sustain BC progression in vitro. Consequently, this study could be a starting point to develop new therapeutic strategies targeting BCAFs and their interactions with cancer cells.

Influence of Tumor Microenvironment and Fibroblast Population Plasticity on Melanoma Growth, Therapy Resistance and Immunoescape.

Cutaneous melanoma (CM) tissue represents a network constituted by cancer cells and tumor microenvironment (TME). A key feature of CM is the high structural and cellular plasticity of TME, allowing its evolution with disease and adaptation to cancer cell and environmental alterations. In particular, during melanoma development and progression each component of TME by interacting with each other and with cancer cells is subjected to dramatic structural and cellular modifications. These alterations affect extracellular matrix (ECM) remodelling, phenotypic profile of stromal cells, cancer growth and therapeutic response. The stromal fibroblast populations of the TME include normal fibroblasts and melanoma-associated fibroblasts (MAFs) that are highly abundant and flexible cell types interacting with melanoma and stromal cells and differently influencing CM outcomes. The shift from the normal microenvironment to TME and from normal fibroblasts to MAFs deeply sustains CM growth. Hence, in this article we review the features of the normal microenvironment and TME and describe the phenotypic plasticity of normal dermal fibroblasts and MAFs, highlighting their roles in normal skin homeostasis and TME regulation. Moreover, we discuss the influence of MAFs and their secretory profiles on TME remodelling, melanoma progression, targeted therapy resistance and immunosurveillance, highlighting the cellular interactions, the signalling pathways and molecules involved in these processes.

A substrate-specific mTORC1 pathway underlies Birt-Hogg-Dubé syndrome.

The mechanistic target of rapamycin complex 1 (mTORC1) is a key metabolic hub that controls the cellular response to environmental cues by exerting its kinase activity on multiple substrates1-3. However, whether mTORC1 responds to diverse stimuli by differentially phosphorylating specific substrates is poorly understood. Here we show that transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy4,5, is phosphorylated by mTORC1 via a substrate-specific mechanism that is mediated by Rag GTPases. Owing to this mechanism, the phosphorylation of TFEB-unlike other substrates of mTORC1, such as S6K and 4E-BP1- is strictly dependent on the amino-acid-mediated activation of RagC and RagD GTPases, but is insensitive to RHEB activity induced by growth factors. This mechanism has a crucial role in Birt-Hogg-Dubé syndrome, a disorder that is caused by mutations in the RagC and RagD activator folliculin (FLCN) and is characterized by benign skin tumours, lung and kidney cysts and renal cell carcinoma6,7. We found that constitutive activation of TFEB is the main driver of the kidney abnormalities and mTORC1 hyperactivity in a mouse model of Birt-Hogg-Dubé syndrome. Accordingly, depletion of TFEB in kidneys of these mice fully rescued the disease phenotype and associated lethality, and normalized mTORC1 activity. Our findings identify a mechanism that enables differential phosphorylation of mTORC1 substrates, the dysregulation of which leads to kidney cysts and cancer.

Shedding light on surface exposition of poly(ethylene glycol) and folate targeting units on nanoparticles of poly(ε-caprolactone) diblock copolymers: Beyond a paradigm.

Polymeric nanoparticles (NPs) of poly(ε-caprolactone) (PCL) covered with a hydrophilic poly(ethylene glycol) (PEG) shell are usually prepared from diblock PEG-PCL copolymers through different techniques. Furthermore PEG, NPs can be decorated with targeting ligands to accumulate in specific cell lines. However, the density and conformation of PEG on the surface and its impact on the exposition of small targeting ligands has been poorly considered so far although this has a huge impact on biological behaviour. Here, we focus on PEG-PCL NPs and their folate-targeted version to encourage accumulation in cancer cells overexpressing folate receptor α. NPs were prepared with mixtures of PEG-PCL with different PEG length (short 1.0kDa, long 2.0kDa,) and a folate-functionalized PEG-PCL (PEG 1.5kDa) by the widely employed solvent displacement method. In depth characterization of NPs surface by 1H NMR, fluorescence and photon correlation spectroscopy evidenced a PEGylation extent below 7% with PEG in a mushroom conformation and the presence of folate more exposed to water pool in the case of copolymer with short PEG. NPs with short PEG adsorbed HSA forming a soft corona without aggregating. Although limited, PEGylation overall reduced NPs uptake in human macrophages. Uptake of NPs exposing folate prepared with short PEG was higher in KB cells (FR+) than in A549 (FR-), occurred via FR-receptor and involved lipid rafts-dependent endocytosis. In conclusion, the present results demonstrate that PEG length critically affects protein interaction and folate exposition with a logical impact on receptor-mediated cell uptake. Our study highlights that the too simplistic view suggesting that PEG-PCL gives PEG-coated NPs needs to be re-examined in the light of actual surface properties, which should always be considered case-by-case.