Background: Triple-negative breast cancer (TNBC) represents the most aggressive breast cancer subtype, which recently attracts great interest for immune therapeutic development. In this context, in-depth understanding of TNBC immune landscape is highly demanded.Results: Here we report full-length single-cell RNA sequencing results of 9683 tumor-infiltrated immune cells isolated from 14 treatment naïve TNBC tumors, where 22 immune cell subsets, including T cells, macrophages, B cells, and DCs have been characterized. We identify a new T cell subset, CD8+CXCL8+ T cell, which associates with poor survival, and a subset of “pre-exhaustion” T cell cluster, which is predictive of favorable prognosis. A novel immune cell subset comprised of TCR+ macrophages, is found to be widely distributed in TNBC tumors. Further analyses reveal an up-regulation of molecules associated with TCR signaling and cytotoxicity in these immune cells.Conclusions: Altogether, our study provides a valuable resource to understand the immune ecosystem of TNBC. The novel immune cell subsets reported herein might be functionally important in cancer immunity. These data will be helpful for the immunotherapeutic strategy design of this disease.

26Triple-negative breast cancer (TNBC) represents the most aggressive breast cancer 27 subtype, which recently attracts great interest for immune therapeutic development. In 28 this context, in-depth understanding of TNBC immune landscape is highly demanded. 29 Here we report single-cell RNA sequencing results of 9683 tumor-infiltrated immune 30 cells isolated from 14 treatment naïve TNBC tumors, where 22 immune cell subsets, 31 including T cells, macrophages, B cells, and DCs have been characterized. We 32 identify a new T cell subset, CD8 + CXCL8 + naïve T cell, which associates with poor 33 survival. A novel immune cell subset comprised of TCR + macrophages, is found to be 34 widely distributed in TNBC tumors. Further analyses reveal an up-regulation of 35 molecules associated with TCR signaling and cytotoxicity in these immune cells, 36 indicating TCR signaling activation. Altogether, our study provides a valuable 37 resource to understand the immune ecosystem of TNBC. The novel immune cell 38 subsets reported herein might be functionally important in cancer immunity. 39 40 SIGNIFICANCE：This work demonstrates a single-cell transcriptome atlas of 41 immune cells in treatment naïve TNBC tumors, revealing novel immune cell subsets. 42This study provides a valuable resource to understand the immune ecosystem of 43 TNBC, which will be helpful for the immunotherapeutic strategy design of TNBC. 44 45 85TNBC tumors are typically more aggressive and difficult to treat than hormone 86 receptor-positive tumors, and are associated with a higher risk of early relapse. The 87 lack of estrogen receptor, progesterone receptor, and HER2 expression precludes the 88 use of targeted therapies, and the only approved systemic treatment option is 89 chemotherapy. Responses to chemotherapy occur, but are often short lived and 90 frequently accompanied by considerable toxicity. Gene profiling studies reveal that 91 TNBCs are highly heterogeneous and a large proportion of them demonstrate DNA 92 Repair Deficiency Signature (18)(19)(20). Recent data showed impressive activity of 93 PD-1/PD-L1 blockade therapy in metastatic TNBC patients who were chemotherapy 94 naïve, suggesting early intervention of immunotherapy can bring more benefit (21). 95Clinical trials applying checkpoint inhibitors in the neo-adjuvant setting of TNBC are 96 ongoing. Although need to be confirmed in a larger cohort, these results are consistent 97 with the notion that immunotherapy agents are most efficient at low tumor burden and 98 in patients naïve of immune-modulatory chemotherapy agents. To better understand 99 the immune ecosystem of TNBC, we analyzed the full-length single-cell RNA 100 sequencing data of 9,683 tumor-infiltrated immune cells isolated from treatment naïve 101 TNBC tumors. We identified 22 unique immune cell subsets, including T cells, 102 macrophages, B cells, and DCs. Using combined expression and TCR-based analyses, 103 we were able to indicate the function and developmental path of T cell subsets. We 104 found a novel T c...

A technical problem of characterizing copy number variation of several cells with next-generation sequencing is the whole genome amplification induced bias. The result of CNVs and mosaicism detection is affected by the GC bias. Here, we report a rapid non-WGA sample preparation strategy for a single-molecule sequencing platform GenoCare1600. This approach, combined with a single-molecule sequencing platform that avoids the use of WGA and bridge PCR processes, can provide higher reliability with its lower GC bias. By combining our optimized Tn5-based transposon insertion approach with GenoCare, we successfully detected CNVs as small as 1.29M and mosaicism as small as 20%, which is consistent with next-generation sequencing (NGS) data. Moreover, our GenoCare-TTI protocol showed less GC bias and less Mad of Diff. These results suggest that the optimized TTI approach, together with the GenoCare1600 sequencing platform, is a promising option for CNV characterization from maybe one single cell.

This study aimed to investigate the effect of fatty acid-ethanol amine (FA-EA) derivatives (L1-L10) on the mitigation of intracellular lipid accumulation and downregulation of pro-inflammatory cytokines in vitro. First, the series of FA-EA derivatives were synthesized and characterized. Then, their cytotoxic, intracellular lipid accumulation and inhibition of pro-inflammatory cytokines were evaluated. The oil red O staining experiment showed that the tested compounds L4, L6, L8, L9, and L10 could reduce intracellular lipid accumulation induced by palmitic acid (PA). Moreover, ω-3/ω-6 PUFA-EA derivatives showed inhibitory effect on the production of pro-inflammatory cytokines in lipopolysaccharide (LPS) -stimulated RAW 264.7 cells. ω-3/ω-6 PUFA-EA derivatives at a concentrations of 10 μM could significantly decrease mRNA levels of IL-6, IL-1β, and TNF-α, inhibit NO production, and alleviate the protein expression of IL-1β in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. These data suggest that ω-3 PUFA-EA derivatives can be beneficial for further pharmaceutical development to treat chronic low-grade inflammation diseases such as obesity.