Chiara Cianciaruso

Background: Although immune checkpoint inhibitors have been a breakthrough in clinical oncology, these therapies fail to produce durable responses in a significant fraction of patients. This lack of long-term efficacy may be due to a poor pre-existing network linking innate and adaptive immunity. Here, we present an antisense oligonucleotide (ASO)-based strategy that dually targets toll-like receptor 9 (TLR9) and programmed cell death ligand 1 (PD-L1), aiming to overcome resistance to anti-PD-L1 monoclonal therapy. Methods: We designed a high-affinity immunomodulatory IM-TLR9:PD-L1-ASO antisense oligonucleotide (hereafter, IM-T9P1-ASO) targeting mouse PD-L1 messenger RNA and activating TLR9. Then, we performed in vitro and in vivo studies to validate the IM-T9P1-ASO activity, efficacy, and biological effects in tumors and draining lymph nodes. We also performed intravital imaging to study IM-T9P1-ASO pharmacokinetics in the tumor. Results: IM-T9P1-ASO therapy, unlike PD-L1 antibody therapy, results in durable antitumor responses in multiple mouse cancer models. Mechanistically, IM-T9P1-ASO activates a state of tumor-associated dendritic cells (DCs), referred to here as DC3s, which have potent antitumor potential but express the PD-L1 checkpoint. IM-T9P1-ASO has two roles: it triggers the expansion of DC3s by engaging with TLR9 and downregulates PD-L1, thereby unleashing the antitumor functions of DC3s. This dual action leads to tumor rejection by T cells. The antitumor efficacy of IM-T9P1-ASO depends on the antitumor cytokine interleukin-12 (IL-12), produced by DC3s, and Batf3, a transcription factor required for DC development. Conclusions: By simultaneously targeting TLR9 and PD-L1, IM-T9P1-ASO amplifies antitumor responses via DC activation, leading to sustained therapeutic efficacy in mice. By highlighting differences and similarities between mouse and human DCs, this study could serve to develop similar therapeutic strategies for patients with cancer.

Lung cancer resistance to PD-1 inhibition can be overcome by targeting tumor-associated macrophages in mouse models.

Biosensors are indispensable tools for public, global, and personalized healthcare as they provide tests that can be used from early disease detection and treatment monitoring to preventing pandemics. We introduce single-wavelength imaging biosensors capable of reconstructing spectral shift information induced by biomarkers dynamically using an advanced data processing technique based on an optimal linear estimator. Our method achieves superior sensitivity without wavelength scanning or spectroscopy instruments. We engineered diatomic dielectric metasurfaces supporting bound states in the continuum that allows high-quality resonances with accessible near-fields by in-plane symmetry breaking. The large-area metasurface chips are configured as microarrays and integrated with microfluidics on an imaging platform for real-time detection of breast cancer extracellular vesicles encompassing exosomes. The optofluidic system has high sensing performance with nearly 70 1/RIU figure-of-merit enabling detection of on average 0.41 nanoparticle/µm² and real-time measurements of extracellular vesicles binding from down to 204 femtomolar solutions. Our biosensors provide the robustness of spectrometric approaches while substituting complex instrumentation with a single-wavelength light source and a complementary-metal-oxide-semiconductor camera, paving the way toward miniaturized devices for point-of-care diagnostics.

Pancreatic beta cells synthesize and secrete the neurotransmitter GABA (γ-aminobutyric acid) as a paracrine and autocrine signal to help regulate hormone secretion and islet homeostasis. Islet GABA release has classically been described as a secretory-vesicle-mediated event. Yet, a limitation of the hypothesized vesicular GABA release from islets is the lack of expression of a vesicular GABA transporter in beta cells. Consequentially, GABA accumulates in the cytosol. Here, we provide evidence that the human beta cell effluxes GABA from a cytosolic pool in a pulsatile manner, imposing a synchronizing rhythm on pulsatile insulin secretion. The volume regulatory anion channel, functionally encoded by LRRC8A or Swell1, is critical for pulsatile GABA secretion. GABA content in beta cells is depleted and secretion is disrupted in islets from patients with type 1 and type 2 diabetes, suggesting that loss of GABA as a synchronizing signal for hormone output may correlate with diabetes pathogenesis.

Cytotoxic chemotherapy is an effective treatment for invasive breast cancer. However, experimental studies in mice also suggest that chemotherapy has pro-metastatic effects. Primary tumours release extracellular vesicles (EVs), including exosomes, that can facilitate the seeding and growth of metastatic cancer cells in distant organs, but the effects of chemotherapy on tumour-derived EVs remain unclear. Here we show that two classes of cytotoxic drugs broadly employed in pre-operative (neoadjuvant) breast cancer therapy, taxanes and anthracyclines, elicit tumour-derived EVs with enhanced pro-metastatic capacity. Chemotherapy-elicited EVs are enriched in annexin A6 (ANXA6), a Ca2+-dependent protein that promotes NF-κB-dependent endothelial cell activation, Ccl2 induction and Ly6C+CCR2+ monocyte expansion in the pulmonary pre-metastatic niche to facilitate the establishment of lung metastasis. Genetic inactivation of Anxa6 in cancer cells or Ccr2 in host cells blunts the pro-metastatic effects of chemotherapy-elicited EVs. ANXA6 is detected, and potentially enriched, in the circulating EVs of breast cancer patients undergoing neoadjuvant chemotherapy.

Chimeric receptors expressed on the surface of antigen-presenting cells enable the uptake of cancer-cell-derived extracellular vesicles and the display of tumor antigens. We describe a lentivirus-encoded chimeric receptor, termed extracellular vesicle (EV)-internalizing receptor (EVIR), which enables the selective uptake of cancer-cell-derived EVs by dendritic cells (DCs). The EVIR enhances DC presentation of EV-associated tumor antigens to CD8+ T cells primarily through MHCI recycling and cross-dressing. EVIRs should facilitate exploring the mechanisms and implications of horizontal transfer of tumor antigens to antigen-presenting cells.

Scientific Reports 7: Article number: 45961; published online: 12 April 2017; updated: 09 May 2017 This Article contains a typographical error in the Methods section under subheading ‘Data Availability. Associated Article’, “Protocol Exchange doi: 1038/Protex.2017.039 (2017)”. should read: “ProtocolExchange doi: 10.

A robust and reproducible method for culturing monolayers of adherent and well-spread primary islet cells on glass coverslips is required for detailed imaging studies by super-resolution and live-cell microscopy. Guided by an observation that dispersed islet cells spread and adhere well on glass surfaces in neuronal co-culture and form a monolayer of connected cells, we demonstrate that in the absence of neurons, well-defined surface coatings combined with components of neuronal culture media collectively support robust attachment and growth of primary human or rat islet cells as monolayers on glass surfaces. The islet cell monolayer cultures on glass stably maintain distinct mono-hormonal insulin+, glucagon+, somatostatin+ and PP+ cells and glucose-responsive synchronized calcium signaling as well as expression of the transcription factors Pdx-1 and NKX-6.1 in beta cells. This technical advance enabled detailed observation of sub-cellular processes in primary human and rat beta cells by super-resolution microscopy. The protocol is envisaged to have broad applicability to sophisticated analyses of pancreatic islet cells that reveal new biological insights, as demonstrated by the identification of an in vitro protocol that markedly increases proliferation of primary beta cells and is associated with a reduction in ciliated, ostensibly proliferation-suppressed beta cells.