umors encompass complex cellular ecosystems of malignant and non-malignant cells, whose diversity and interactions affect cancer progression and drug response and resistance. Recent advances in single-cell genomics, especially single-cell RNA-Seq (scRNA-Seq), have transformed our ability to analyze tumors, revealing cell types, states, genetic diversity and interactions in the complex tumor ecosystem 1-6. Single-cell analysis of tumors is rapidly expanding, including the launch of a Human Tumor Atlas Network (HTAPP) as part of the Cancer Moonshot 7. Successful scRNA-Seq of clinical tumor specimens poses several challenges. First, it requires quick dissociation tailored to the tumor type, and involves enzymatic digestion, which can lead to loss of sensitive cells or changes in gene expression. Moreover, obtaining fresh tissue is time-sensitive and requires tight coordination

AbstractSingle cell genomics is essential to chart the complex tumor ecosystem. While single cell RNA-Seq (scRNA-Seq) profiles RNA from cells dissociated from fresh tumor tissues, single nucleus RNA-Seq (snRNA-Seq) is needed to profile frozen or hard-to-dissociate tumors. Each strategy requires modifications to fit the unique characteristics of different tissue and tumor types, posing a barrier to adoption. Here, we developed a systematic toolbox for profiling fresh and frozen clinical tumor samples using scRNA-Seq and snRNA-Seq, respectively. We tested eight tumor types of varying tissue and sample characteristics (resection, biopsy, ascites, and orthotopic patient-derived xenograft): lung cancer, metastatic breast cancer, ovarian cancer, melanoma, neuroblastoma, pediatric sarcoma, glioblastoma, pediatric high-grade glioma, and chronic lymphocytic leukemia. Analyzing 212,498 cells and nuclei from 39 clinical samples, we evaluated protocols by cell quality, recovery rate, and cellular composition. We optimized protocols for fresh tissue dissociation for different tumor types using a decision tree to account for the technical and biological variation between clinical samples. We established methods for nucleus isolation from OCT embedded and fresh-frozen tissues, with an optimization matrix varying mechanical force, buffer, and detergent. scRNA-Seq and snRNA-Seq from matched samples recovered the same cell types and intrinsic expression profiles, but at different proportions. Our work provides direct guidance across a broad range of tumors, including criteria for testing and selecting methods from the toolbox for other tumors, thus paving the way for charting tumor atlases.

Pneumonia as a secondary infection after influenza is a major cause of excess morbidity and mortality despite the availability and use of antibiotics active against Streptococcus pneumoniae. We hypothesized that use of a bacteriostatic protein synthesis inhibitor would improve outcomes by reducing the inflammatory response. BALB/c mice infected with influenza virus and super-infected with Streptococcus pneumoniae were treated with either the cell wall active antibiotic ampicillin or protein synthesis inhibitors clindamycin or azithromycin. Ampicillin therapy performed significantly worse (56% survival) in the model compared to clindamycin therapy either alone (82%) or in combination with ampicillin (80%) and to azithromycin (92%). Improved survival appeared to be mediated by decreased inflammation manifested as lower levels of inflammatory cells and pro-inflammatory cytokines in the lungs, and less severe histopathology. These data suggest that beta-lactam therapy may not be optimal as first line treatment of community acquired pneumonia when it follows influenza.

Host inflammatory responses contribute to the significant immunopathology that occurs during treatment of secondary bacterial pneumonia following influenza. We undertook the present study to determine the mechanisms underlying disparate outcomes in a mouse model with β-lactam and macrolide antibiotics. Lysis of superinfecting bacteria by ampicillin caused an extensive influx of neutrophils into the lungs resulting in a consolidative pneumonia, necrotic lung damage, and significant mortality. This was mediated through Toll-like receptor (TLR) 2 and was independent of TLR4 and the Streptococcus pneumoniae cytotoxin pneumolysin. Treatment with azithromycin prevented neutrophil accumulation and rescued mice from subsequent mortality. This effect was independent of the antibacterial activity of this macrolide since dual therapy with ampicillin and azithromycin against an azithromycin-resistant strain also was able to cure secondary pneumonia. These data suggest that strategies for eliminating bacteria without lysis coupled with immunomodulation of inflammation should be pursued clinically.

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