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Collagen-producing cells maintain the complex architecture of the lung and drive pathologic scarring in pulmonary fibrosis. Here we perform single-cell RNA-sequencing to identify all collagen-producing cells in normal and fibrotic lungs. We characterize multiple collagen-producing subpopulations with distinct anatomical localizations in different compartments of murine lungs. One subpopulation, characterized by expression of Cthrc1 (collagen triple helix repeat containing 1), emerges in fibrotic lungs and expresses the highest levels of collagens. Single-cell RNA-sequencing of human lungs, including those from idiopathic pulmonary fibrosis and scleroderma patients, demonstrate similar heterogeneity and CTHRC1 -expressing fibroblasts present uniquely in fibrotic lungs. Immunostaining and in situ hybridization show that these cells are concentrated within fibroblastic foci. We purify collagen-producing subpopulations and find disease-relevant phenotypes of Cthrc1 -expressing fibroblasts in in vitro and adoptive transfer experiments. Our atlas of collagen-producing cells provides a roadmap for studying the roles of these unique populations in homeostasis and pathologic fibrosis. Collagen production by lung cells is critical to maintain organ architecture but can also drive pathological scarring. Here the authors perform single cell RNA sequencing of collagen-producing lung cells identifying a subset of pathologic fibroblasts characterized by Cthrc1 expression which are concentrated within fibroblastic foci in fibrotic lungs and show a pro-fibrotic phenotype.

α-Smooth muscle actin (α-SMA) is used as a marker for a subset of activated fibrogenic cells, myofibroblasts, which are regarded as important effector cells of tissue fibrogenesis. We address whether α-SMA-expressing myofibroblasts are detectable in fibrotic muscles of mdx5cv mice, a mouse model for Duchenne muscular dystrophy (DMD), and whether the α-SMA expression correlates with the fibrogenic function of intramuscular fibrogenic cells. α-SMA immunostaining signal was not detected in collagen I (GFP)-expressing cells in fibrotic muscles of ColI-GFP/mdx5cv mice, but it was readily detected in smooth muscle cells lining intramuscular blood vessel walls. α-SMA expression was detected by quantitative RT-PCR and Western blot in fibrogenic cells sorted from diaphragm and quadriceps muscles of the ColI-GFP/mdx5cv mice. Consistent with the more severe fibrosis in the ColI-GFP/mdx5cv diaphragm, the fibrogenic cells in the diaphragm exerted a stronger fibrogenic function than the fibrogenic cells in the quadriceps as gauged by their extracellular matrix gene expression. However, both gene and protein expression of α-SMA was lower in the diaphragm fibrogenic cells than in the quadriceps fibrogenic cells in the ColI-GFP/mdx5cv mice. We conclude that myofibroblasts are present in fibrotic skeletal muscles, but their expression of α-SMA is not detectable by immunostaining. The level of α-SMA expression by intramuscular fibrogenic cells does not correlate positively with the level of collagen gene expression or the severity of skeletal muscle fibrosis in the mdx5cv mice. α-SMA is not a functional marker of fibrogenic cells in skeletal muscle fibrosis associated with muscular dystrophy.

Compromised regenerative capacity of lung epithelial cells can lead to cellular senescence, which may precipitate fibrosis. While increased markers of senescence have been reported in idiopathic pulmonary fibrosis (IPF), the origin and identity of these senescent cells remain unclear, and tools to characterize context-specific cellular senescence in human lung are lacking. We observed that the senescent marker p16 is predominantly localized to bronchiolized epithelial structures in scarred regions of IPF and systemic sclerosis-associated interstitial lung disease (SSc-ILD) lung tissue, overlapping with the basal epithelial markers Keratin 5 and Keratin 17. Using in vitro models, we derived transcriptional signatures of senescence programming specific to different types of lung epithelial cells and interrogated these signatures in a single-cell RNA-Seq data set derived from control, IPF, and SSc-ILD lung tissue. We identified a population of basal epithelial cells defined by, and enriched for, markers of cellular senescence and identified candidate markers specific to senescent basal epithelial cells in ILD that can enable future functional studies. Notably, gene expression of these cells significantly overlaps with terminally differentiating cells in stratified epithelia, where it is driven by p53 activation as part of the senescence program.

We developed an automated high-throughput Smart-seq3 (HT Smart-seq3) workflow that integrates best practices and an optimized protocol to enhance efficiency, scalability, and method reproducibility. This workflow consistently produces high-quality data with high cell capture efficiency and gene detection sensitivity. In a rigorous comparison with the 10X platform using human primary CD4 + T-cells, HT Smart-seq3 demonstrated higher cell capture efficiency, greater gene detection sensitivity, and lower dropout rates. Additionally, when sufficiently scaled, HT Smart-seq3 achieved a comparable resolution of cellular heterogeneity to 10X. Notably, through T-cell receptor (TCR) reconstruction, HT Smart-seq3 identified a greater number of productive alpha and beta chain pairs without the need for additional primer design to amplify full-length V(D)J segments, enabling more comprehensive TCR profiling across a broader range of species. Taken together, HT Smart-seq3 overcomes key technical challenges, offering distinct advantages that position it as a promising solution for the characterization of single-cell transcriptomes and immune repertoires, particularly well-suited for low-input, low-RNA content samples.

CD47 is a \"don't eat me\" signal that is overexpressed in tumors to evade phagocytosis by tumor associated macrophages (TAM). Investigational agents targeting CD47, such as magrolimab, aim to induce phagocytosis of tumor cells by TAMs. Previously, two key TAM subsets have been identified: C1QC TAMs, which display pro-phagocytic activity, and SPP1 TAMs that express pro-angiogenic markers. We characterize CD47 expression and its relationship with tumor macrophages in solid tumor samples. Resectable tumors from head and neck squamous cell carcinoma (n=36) (HNSCC), breast cancer (n=37) (BC), and colorectal cancer (n=36) (CRC) were evaluated for CD47 expression by immunohistochemistry (IHC), two multiplex immunofluorescence panels were used to characterize TAM markers and T cell markers. RNA sequencing was also performed. CD47 protein expression was higher on tumor cells compared to stromal cells across tumor indications tested. Although CRC had the lowest prevalence for CD47 expression in primary tumors, we observed a marked increase in CD47 expression in CRC liver metastases. We developed an SPP1 TAM gene signature and validated a C1QC TAM gene signature to estimate TAM abundances from bulk RNA-Seq data. In the TAM mIF analysis, HNSCC had the highest macrophage density of the indications tested. We observed a positive correlation between a higher C1QC: SPP1 TAM ratio and macrophage phenotype and tumor T cell density. C1QC macrophage signatures correlate with tumor CD47 protein expression in BC and HNSCC samples, suggesting interplay between them. We characterized CD47 expression across key solid tumor indications being evaluated clinically using anti-CD47 blockade agents: HNSCC, breast cancer and CRC. Using a CD47 IHC assay, we identified HNSCC as an indication with the highest CD47 expression. In addition, we quantified tumor macrophages using multiplex immunofluorescence (mIF) and determined that HNSCC had the highest density of TAMs. Compared to relatively low CD47 expression in primary CRC tumors, CRC liver metastases had very high CD47 expression. Quantification of TAM signatures and CD47 expression represent key biomarkers to monitor in patient samples during exploration of CD47-blockade agents in the clinic.

BackgroundImmune checkpoint blockade targeting the PD-1/PD-L1 axis has revolutionized the treatment of many solid tumors. PD-L1 expression is known to be modulated by both infiltrating T cells and by tumor intrinsic mechanisms. Therapeutics targeting the macrophage checkpoints CD47 and SIRPα are now being evaluated clinically, but little is known about how CD47 expression is influenced by tumor molecular subtypes or the presence of immune cells, particularly macrophages.MethodsFormalin fixed and paraffin embedded samples of primary, resectable tumors from patients with HNSCC (N=44), TNBC (N=48) and CRC (N=48) were commercially procured and evaluated for CD47 (Abcam clone EPR21794) and SIRPα (LSBio LS-B551) expression by IHC. DNA/RNA were extracted from all samples and subjected to RNA-Seq and whole exome sequencing (WES). Multiplex immunofluorescence (mIF) was performed on 26 samples from each indication utilizing two custom 4-plex immunofluorescence assays. A macrophage panel evaluated CD68, CD163, PD-L1 and cytokeratin and a tissue resident memory T cell panel evaluated CD3, CD8, CD103 and cytokeratin.ResultsCD47 expression was upregulated in the tumor versus the surrounding stroma in all three indications and was highest in HNSCC, followed by TNBC and CRC (figure 1). CD47 expression as measured by IHC was used to subdivide samples into high and low CD47 expression categories using the median within each indication. A phagocytic tumor associated macrophage (TAM) gene signature1 was significantly higher in the high CD47 samples from HNSCC and CRC (figure 1). Characterization of macrophages (mIF panel) indicated that CD163+/CD68- macrophages were the most abundant macrophages in all three indications, demonstrated increased prevalence within the tumor stroma, and correlated strongly with the phagocytic TAM gene signature. PD-L1 expression was predominantly found on CD68+/CD163- macrophages in all tumor indications. We classified samples using molecular subtyping for CRC,2 and unsupervised clustering for HNSCC and TNBC. CD47 expression was generally not differentiated across the CRC molecular subtypes.ConclusionsCD47 targeting therapies are currently being evaluated in clinical trials in multiple solid tumor indications. Here were demonstrate that CD47 expression is higher in tumor compared to stroma in HNSCC, TNBC and CRC and correlates with an increased phagocytic TAM signature. These data suggest that some tumors may avoid immune surveillance by upregulating CD47 in the presence of phagocytic macrophages, and that tumors with these characteristics could be rationale candidates for evaluation of CD47 based therapies.ReferencesZhang L, Li Z, Skrzypczynska KM, Fang Q, Zhang W, O’Brien SA, He Y, Wang L, Zhang Q, Kim A et al. Single-Cell Analyses Inform Mechanisms of Myeloid-Targeted Therapies in Colon Cancer. Cell 2020, 181(2):442–459 e429.Dienstmann R, Vermeulen L, Guinney J, Kopetz S, Tejpar S, Tabernero J: Consensus molecular subtypes and the evolution of precision medicine in colorectal cancer. Nat Rev Cancer 2017, 17(2):79–92.Abstract 515 Figure 1

Respiratory syncytial virus (RSV) is a significant cause of morbidity and mortality in high-risk populations. Although prophylactic options are available, there are no effective oral therapeutics for RSV infection. Obeldesivir (ODV) is an orally bioavailable prodrug of the nucleoside analog GS-441524, which is converted intracellularly to its active nucleoside triphosphate and inhibits the RSV RNA polymerase. Here we report the potent antiviral activity of ODV against geographically and temporally diverse RSV A and B clinical isolates (EC 50 : 0.20–0.66 μM). Resistance selection studies with ODV and GS-441524 against RSV identify a single amino acid substitution, I777L, in the L polymerase with reduced susceptibility (3.3-3.8-fold) to ODV and GS-441524, indicating a high barrier for resistance development. In an African green monkey RSV infection model, once-daily oral ODV doses of 30 or 90 mg/kg initiated ~24 hours post-infection significantly reduces log 10 viral RNA copies/mL × day area under the curve by 69–92% in the upper and lower respiratory tracts. Together, these preclinical data support the clinical evaluation of ODV for the treatment of RSV infection. In this work, authors show that the nucleoside prodrug obeldesivir has potent antiviral activity across respiratory syncytial virus (RSV) clinical isolates with a high resistance barrier. Once-daily obeldesivir treatment was efficacious against RSV in a non-human primate model.

[alpha]-Smooth muscle actin ([alpha]-SMA) is used as a marker for a subset of activated fibrogenic cells, myofibroblasts, which are regarded as important effector cells of tissue fibrogenesis. We address whether [alpha]-SMA-expressing myofibroblasts are detectable in fibrotic muscles of mdx.sup.5cv mice, a mouse model for Duchenne muscular dystrophy (DMD), and whether the [alpha]-SMA expression correlates with the fibrogenic function of intramuscular fibrogenic cells. [alpha]-SMA immunostaining signal was not detected in collagen I (GFP)-expressing cells in fibrotic muscles of ColI-GFP/mdx.sup.5cv mice, but it was readily detected in smooth muscle cells lining intramuscular blood vessel walls. [alpha]-SMA expression was detected by quantitative RT-PCR and Western blot in fibrogenic cells sorted from diaphragm and quadriceps muscles of the ColI-GFP/mdx.sup.5cv mice. Consistent with the more severe fibrosis in the ColI-GFP/mdx.sup.5cv diaphragm, the fibrogenic cells in the diaphragm exerted a stronger fibrogenic function than the fibrogenic cells in the quadriceps as gauged by their extracellular matrix gene expression. However, both gene and protein expression of [alpha]-SMA was lower in the diaphragm fibrogenic cells than in the quadriceps fibrogenic cells in the ColI-GFP/mdx.sup.5cv mice. We conclude that myofibroblasts are present in fibrotic skeletal muscles, but their expression of [alpha]-SMA is not detectable by immunostaining. The level of [alpha]-SMA expression by intramuscular fibrogenic cells does not correlate positively with the level of collagen gene expression or the severity of skeletal muscle fibrosis in the mdx.sup.5cv mice. [alpha]-SMA is not a functional marker of fibrogenic cells in skeletal muscle fibrosis associated with muscular dystrophy.

Respiratory syncytial virus (RSV) is a significant cause of morbidity and mortality in high-risk populations. Although prophylactic options are available, there are no effective oral therapeutics for RSV infection. Obeldesivir (ODV) is an orally bioavailable prodrug of the nucleoside analog GS-441524, which is converted intracellularly to its active nucleoside triphosphate and inhibits the RSV RNA polymerase. Here we report the potent antiviral activity of ODV against geographically and temporally diverse RSV A and B clinical isolates (EC50: 0.20-0.66 μM). Resistance selection studies with ODV and GS-441524 against RSV identified a single amino acid substitution, I777L, in the L polymerase with reduced susceptibility (3.3-3.8-fold) to ODV and GS-441524, indicating a high barrier for resistance development. In an African green monkey RSV infection model, once-daily oral ODV doses of 30 or 90 mg/kg initiated ~24 hours post-infection significantly reduced log10 viral RNA copies/mL×day area under the curve by 69-92% in the upper and lower respiratory tracts. Together, these preclinical data support the clinical evaluation of ODV for the treatment of RSV infection.Competing Interest StatementAll authors affiliated with Gilead Sciences may hold stock or stock options in Gilead Sciences, Inc. VA, PAP, KS, and PLD received funding from Gilead Sciences Inc. to support parts of this work. PAP received grant awards and ad hoc honoraria from Merck and Shionogi for consulting and scientific boards unrelated to the work presented here. All other authors declare no competing interests.