A multiparameter liquid biopsy approach allows to track melanoma dynamics and identify early treatment resistance.

Melanoma heterogeneity is a hurdle in metastatic disease management. Although the advent of targeted therapy has significantly improved patient outcomes, the occurrence of resistance makes monitoring of the tumor genetic landscape mandatory. Liquid biopsy could represent an important biomarker for the real-time tracing of disease evolution. Thus, we aimed to correlate liquid biopsy dynamics with treatment response and progression by devising a multiplatform approach applied to longitudinal melanoma patient monitoring. We conceived an approach that exploits Next Generation Sequencing (NGS) and droplet digital PCR, as well as the FDA-cleared platform CellSearch, to analyze circulating tumor DNA (ctDNA) trend and circulating melanoma cell (CMC) count, together with their customized genetic and copy number variation analysis. The approach was applied to 17 stage IV melanoma patients treated with BRAF/MEK inhibitors, followed for up to 28 months. BRAF mutations were detected in the plasma of 82% of patients. Single nucleotide variants known or suspected to confer resistance were identified in 70% of patients. Moreover, the amount of ctDNA, both at baseline and during response, correlated with the type and duration of the response itself, and the CMC count was confirmed to be a prognostic biomarker. This work provides proof of principle of the power of this approach and paves the way for a validation study aimed at evaluating early ctDNA-guided treatment decisions in stage IV melanoma. The NGS-based molecular profile complemented the analysis of ctDNA trend and, together with CMC analysis, revealed to be useful in capturing tumor evolution.

Disabled Homolog 2 Controls Prometastatic Activity of Tumor-Associated Macrophages.

Tumor-associated macrophages (TAM) are regulators of extracellular matrix (ECM) remodeling and metastatic progression, the main cause of cancer-associated death. We found that disabled homolog 2 mitogen-responsive phosphoprotein (DAB2) is highly expressed in tumor-infiltrating TAMs and that its genetic ablation significantly impairs lung metastasis formation. DAB2-expressing TAMs, mainly localized along the tumor-invasive front, participate in integrin recycling, ECM remodeling, and directional migration in a tridimensional matrix. DAB2+ macrophages escort the invasive dissemination of cancer cells by a mechanosensing pathway requiring the transcription factor YAP. In human lobular breast and gastric carcinomas, DAB2+ TAMs correlated with a poor clinical outcome, identifying DAB2 as potential prognostic biomarker for stratification of patients with cancer. DAB2 is therefore central for the prometastatic activity of TAMs. SIGNIFICANCE: DAB2 expression in macrophages is essential for metastasis formation but not primary tumor growth. Mechanosensing cues, activating the complex YAP-TAZ, regulate DAB2 in macrophages, which in turn controls integrin recycling and ECM remodeling in 3-D tissue matrix. The presence of DAB2+ TAMs in patients with cancer correlates with worse prognosis.This article is highlighted in the In This Issue feature, p. 1611.

A juxtacrine/paracrine loop between C-Kit and stem cell factor promotes cancer stem cell survival in epithelial ovarian cancer.

Receptors tyrosine kinase (RTK) enable normal and tumor cells to perceive and adapt to stimuli present in the microenvironment. These stimuli, also known as growth factors, are important molecular cues actively supporting cancer stem cell (CSC) self-renewal and viability. Since in epithelial ovarian cancer (EOC) the expression of c-Kit (CD117) has been identified as a CSC hallmark, we investigated the existence of a tumor growth-promoting loop between c-Kit and its ligand Stem Cell Factor (SCF). SCF exists as a soluble or transmembrane protein and through c-Kit interaction regulates cell viability, proliferation, and differentiation both in physiological and pathological conditions. High amounts of SCF were found in the ascitic effusions collected from EOC patients. While tumor cells and CSC only expressed the membrane-associated SCF isoform, both secreted and membrane-bound isoforms were expressed by tumor-associated macrophages (TAM, here shown to be M2-like) and fibroblasts (TAF). Circulating monocytes from EOC-bearing patients and healthy donors did not express both SCF isoforms. However, monocytes isolated from healthy donors produced SCF upon in vitro differentiation into macrophages, irrespectively of M1 or M2 polarization. In vitro, both SCF isoforms were able to activate the Akt pathway in c-Kit+ cells, and this effect was counteracted by the tyrosine kinase inhibitor imatinib. In addition, our results indicated that SCF could help c-Kit+ CSC survival in selective culture conditions and promote their canonical stemness properties, thus indicating the possible existence of a juxtacrine/paracrine circuit in EOC.

Protamine nanocapsules as carriers for oral peptide delivery.

Peptides represent a promising therapeutic class with the potential to alleviate many severe diseases. A key limitation of these active molecules relies on the difficulties for their efficient oral administration. The objective of this work has been the rational design of polymer nanocapsules (NCs) intended for the oral delivery of peptide drugs. For this purpose, we selected insulin glulisine as a model peptide. The polymer shell of the NCs was made of a single layer of protamine, a cationic polypeptide selected for its cell penetration properties, or a double protamine/polysialic acid (PSA) layer. Insulin glulisine-loaded protamine and protamine/PSA NCs, prepared by the solvent displacement method, exhibited a size that varied in the range of 200-400 nm and a neutral surface charge (from +8 mV to -6 mV), depending on the formulation. The stability of the encapsulated peptide was assessed using circular dichroism and an in vitro cell activity study. Colloidal stability studies were also performed in simulated intestinal media containing enzymes and the results indicated that protamine NCs were stable and able to protect insulin from the harsh intestinal environment, and that this capacity could be further enhanced with a double PSA-Protamine layer. These NCs were freeze-dried and stored at room temperature without alteration of the physicochemical properties. When the insulin-loaded protamine NCs were administered intra-intestinally to diabetic rats (12 h fasting) it resulted in a prolonged glucose reduction (60%) as compared to the control insulin solution. This work raises prospects that protamine NCs may have a potential as oral peptide delivery nanocarriers.

The Interplay between Circulating Tumor Cells and the Immune System: From Immune Escape to Cancer Immunotherapy.

Circulating tumor cells (CTCs) have aroused increasing interest not only in mechanistic studies of metastasis, but also for translational applications, such as patient monitoring, treatment choice, and treatment change due to tumor resistance. In this review, we will assess the state of the art about the study of the interactions between CTCs and the immune system. We intend to analyze the impact that the cells of the immune system have in limiting or promoting the metastatic capability of CTCs. To this purpose, we will examine studies that correlate CTCs, immune cells, and patient prognosis, and we will also discuss relevant animal models that have contributed to the understanding of the mechanisms of immune-mediated metastasis. We will then consider some studies in which CTCs seem to play a promising role in monitoring cancer patients during immunotherapy regimens. We believe that, from an accurate and profound knowledge of the interactions between CTCs and the immune system, new immunotherapeutic strategies against cancer might emerge in the future.

4PD Functionalized Dendrimers: A Flexible Tool for In Vivo Gene Silencing of Tumor-Educated Myeloid Cells.

Myeloid cells play a key role in tumor progression and metastasis by providing nourishment and immune protection, as well as facilitating cancer invasion and seeding to distal sites. Although advances have been made in understanding the biology of these tumor-educated myeloid cells (TEMCs), their intrinsic plasticity challenges our further understanding of their biology. Indeed, in vitro experiments only mimic the in vivo setting, and current gene-knockout technologies do not allow the simultaneous, temporally controlled, and cell-specific silencing of multiple genes or pathways. In this article, we describe the 4PD nanoplatform, which allows the in vivo preferential transfection and in vivo tracking of TEMCs with the desired RNAs. This platform is based on the conjugation of CD124/IL-4Rα-targeting peptide with G5 PAMAM dendrimers as the loading surface and can convey therapeutic or experimental RNAs of interest. When injected i.v. in mice bearing CT26 colon carcinoma or B16 melanoma, the 4PD nanoparticles predominantly accumulate at the tumor site, transfecting intratumoral myeloid cells. The use of 4PD to deliver a combination of STAT3- and C/EBPß-specific short hairpin RNA or miR-142-3p confirmed the importance of these genes and microRNAs in TEMC biology and indicates that silencing of both genes is necessary to increase the efficacy of immune interventions. Thus, the 4PD nanoparticle can rapidly and cost effectively modulate and assess the in vivo function of microRNAs and mRNAs in TEMCs.