Anti-tumor activity of a novel LAIR1 antagonist in combination with anti-PD-1 to treat collagen-rich solid tumors.

We recently reported that resistance to PD-1-blockade in a refractory lung cancer-derived model involved increased collagen deposition and the collagen-binding inhibitory receptor leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), and thus we hypothesized that LAIR1 and collagen cooperated to suppress therapeutic response. Here, we report LAIR1 is associated with tumor stroma and is highly expressed by intratumoral myeloid cells in both human tumors and mouse models of cancer. Stroma-associated myeloid cells exhibit a suppressive phenotype and correlate with LAIR1 expression in human cancer. NGM438, a novel humanized LAIR1 antagonist monoclonal antibody, elicits myeloid inflammation and allogeneic T cell responses by binding to LAIR1 and blocking collagen engagement. Further, a mouse-reactive NGM438 surrogate antibody sensitized refractory KP mouse lung tumors to anti-PD-1 therapy and resulted in increased intratumoral CD8+ T cell content and inflammatory gene expression. These data place LAIR1 at the intersection of stroma and suppressive myeloid cells and support the notion that blockade of the LAIR1/collagen axis can potentially address resistance to checkpoint inhibitor therapy in the clinic.

ILT2 and ILT4 Drive Myeloid Suppression via Both Overlapping and Distinct Mechanisms.

Solid tumors are dense three-dimensional (3D) multicellular structures that enable efficient receptor-ligand trans interactions via close cell-cell contact. Immunoglobulin-like transcript (ILT)2 and ILT4 are related immune-suppressive receptors that play a role in the inhibition of myeloid cells within the tumor microenvironment. The relative contribution of ILT2 and ILT4 to immune inhibition in the context of solid tumor tissue has not been fully explored. We present evidence that both ILT2 and ILT4 contribute to myeloid inhibition. We found that although ILT2 inhibits myeloid cell activation in the context of trans-engagement by MHC-I, ILT4 efficiently inhibits myeloid cells in the presence of either cis- or trans-engagement. In a 3D spheroid tumor model, dual ILT2/ILT4 blockade was required for the optimal activation of myeloid cells, including the secretion of CXCL9 and CCL5, upregulation of CD86 on dendritic cells, and downregulation of CD163 on macrophages. Humanized mouse tumor models showed increased immune activation and cytolytic T-cell activity with combined ILT2 and ILT4 blockade, including evidence of the generation of immune niches, which have been shown to correlate with clinical response to immune-checkpoint blockade. In a human tumor explant histoculture system, dual ILT2/ILT4 blockade increased CXCL9 secretion, downregulated CD163 expression, and increased the expression of M1 macrophage, IFNγ, and cytolytic T-cell gene signatures. Thus, we have revealed distinct contributions of ILT2 and ILT4 to myeloid cell biology and provide proof-of-concept data supporting the combined blockade of ILT2 and ILT4 to therapeutically induce optimal myeloid cell reprogramming in the tumor microenvironment.

Extracellular microRNA 3' end modification across diverse body fluids.

microRNAs (miRNAs) are small non-coding RNAs that play critical roles in gene regulation. The presence of miRNAs in extracellular biofluids is increasingly recognized. However, most previous characterization of extracellular miRNAs focused on their overall expression levels. Alternative sequence isoforms and modifications of miRNAs were rarely considered in the extracellular space. Here, we developed a highly accurate bioinformatic method, called miNTA, to identify 3' non-templated additions (NTAs) of miRNAs using small RNA-sequencing data. Using miNTA, we conducted an in-depth analysis of miRNA 3' NTA profiles in 1047 extracellular RNA-sequencing data sets of 4 types of biofluids. This analysis identified hundreds of miRNAs with 3' uridylation or adenylation, with the former being more prevalent. Among these miRNAs, up to 53% (22%) had an average 3' uridylation (adenylation) level of at least 10% in a specific biofluid. Strikingly, we found that 3' uridylation levels enabled segregation of different types of biofluids, more effectively than overall miRNA expression levels. This observation suggests that 3' NTA levels possess fluid-specific information relatively robust to batch effects. In addition, we observed that extracellular miRNAs with 3' uridylations are enriched in processes related to angiogenesis, apoptosis, and inflammatory response, and this type of modification may stabilize base-pairing between miRNAs and their target genes. Together, our study provides a comprehensive landscape of miRNA NTAs in human biofluids, which paves way for further biomarker discoveries. The insights generated in our work built a foundation for future functional, mechanistic, and translational discoveries.

Molecular consequences of fetal alcohol exposure on amniotic exosomal miRNAs with functional implications for stem cell potency and differentiation.

Alcohol (ethanol, EtOH) consumption during pregnancy can result in fetal alcohol spectrum disorders (FASDs), which are characterized by prenatal and postnatal growth restriction and craniofacial dysmorphology. Recently, cell-derived extracellular vesicles, including exosomes and microvesicles containing several species of RNAs (exRNAs), have emerged as a mechanism of cell-to-cell communication. However, EtOH's effects on the biogenesis and function of non-coding exRNAs during fetal development have not been explored. Therefore, we studied the effects of maternal EtOH exposure on the composition of exosomal RNAs in the amniotic fluid (AF) using rat fetal alcohol exposure (FAE) model. Through RNA-Seq analysis we identified and verified AF exosomal miRNAs with differential expression levels specifically associated with maternal EtOH exposure. Uptake of purified FAE AF exosomes by rBMSCs resulted in significant alteration of molecular markers associated with osteogenic differentiation of rBMSCs. We also determined putative functional roles for AF exosomal miRNAs (miR-199a-3p, miR-214-3p and let-7g) that are dysregulated by FAE in osteogenic differentiation of rBMSCs. Our results demonstrate that FAE alters AF exosomal miRNAs and that exosomal transfer of dysregulated miRNAs has significant molecular effects on stem cell regulation and differentiation. Our results further suggest the usefulness of assessing molecular alterations in AF exRNAs to study the mechanisms of FAE teratogenesis that should be further investigated by using an in vivo model.

Characterization of Human Salivary Extracellular RNA by Next-generation Sequencing.

BACKGROUND: It was recently discovered that abundant and stable extracellular RNA (exRNA) species exist in bodily fluids. Saliva is an emerging biofluid for biomarker development for noninvasive detection and screening of local and systemic diseases. Use of RNA-Sequencing (RNA-Seq) to profile exRNA is rapidly growing; however, no single preparation and analysis protocol can be used for all biofluids. Specifically, RNA-Seq of saliva is particularly challenging owing to high abundance of bacterial contents and low abundance of salivary exRNA. Given the laborious procedures needed for RNA-Seq library construction, sequencing, data storage, and data analysis, saliva-specific and optimized protocols are essential. METHODS: We compared different RNA isolation methods and library construction kits for long and small RNA sequencing. The role of ribosomal RNA (rRNA) depletion also was evaluated. RESULTS: The miRNeasy Micro Kit (Qiagen) showed the highest total RNA yield (70.8 ng/mL cell-free saliva) and best small RNA recovery, and the NEBNext library preparation kits resulted in the highest number of detected human genes [5649-6813 at 1 reads per kilobase RNA per million mapped (RPKM)] and small RNAs [482-696 microRNAs (miRNAs) and 190-214 other small RNAs]. The proportion of human RNA-Seq reads was much higher in rRNA-depleted saliva samples (41%) than in samples without rRNA depletion (14%). In addition, the transfer RNA (tRNA)-derived RNA fragments (tRFs), a novel class of small RNAs, were highly abundant in human saliva, specifically tRF-4 (4%) and tRF-5 (15.25%). CONCLUSIONS: Our results may help in selection of the best adapted methods of RNA isolation and small and long RNA library constructions for salivary exRNA studies.

Novel approaches for bioinformatic analysis of salivary RNA sequencing data for development.

Motivation: Analysis of RNA sequencing (RNA-Seq) data in human saliva is challenging. Lack of standardization and unification of the bioinformatic procedures undermines saliva's diagnostic potential. Thus, it motivated us to perform this study. Results: We applied principal pipelines for bioinformatic analysis of small RNA-Seq data of saliva of 98 healthy Korean volunteers including either direct or indirect mapping of the reads to the human genome using Bowtie1. Analysis of alignments to exogenous genomes by another pipeline revealed that almost all of the reads map to bacterial genomes. Thus, salivary exRNA has fundamental properties that warrant the design of unique additional steps while performing the bioinformatic analysis. Our pipelines can serve as potential guidelines for processing of RNA-Seq data of human saliva. Availability and implementation: Processing and analysis results of the experimental data generated by the exceRpt (v4.6.3) small RNA-seq pipeline (github.gersteinlab.org/exceRpt) are available from exRNA atlas (exrna-atlas.org). Alignment to exogenous genomes and their quantification results were used in this paper for the analyses of small RNAs of exogenous origin. Contact: dtww@ucla.edu.