Tobacco use is linked to multiple cancer types and a quarter of cancer deaths. Tobacco smoke contains numerous mutagenic chemicals such as polycyclic aromatic hydrocarbons (PAH), however, the mutagenicity of many chemicals in tobacco products remains underexplored. The tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N’-nitrosonornicotine (NNN), both International Agency for Research on Cancer (IARC) Group 1 carcinogens, form pre-mutagenic pyridyloxobutyl (POB) DNA adducts, yet little is known about the associated genomic mutation landscape(s) in humans. We applied bulk and error-corrected next-generation sequencing to a battery of experimental systems comprising mammalian cell lines and animals exposed to NNK or NNN, and the extracted mutational signatures were compared to POB DNA adducts formed in the same models. A genome-scale POB mutational signature was extracted and characterized in detail and subsequently used for optimized per-sample signature attribution in 6,670 cancer genomes from the International Cancer Genome Consortium Pan-Cancer Analysis of Whole Genomes (ICGC PCAWG) and The Cancer Genome Atlas (TCGA) collections. POB signatures generated in experimental exposure models share profiles dominated by T > N and C > T alterations, with transcription strand asymmetry indicative of damage on thymidines and cytidines. The T > N-dominated mutation profiles can be explained by O2-POB-dT adducts, detected in the exposed cells. Stringent screening of 6,668 cancer genomes revealed the presence of a genome-scale POB signature in 350 tumors from sites associated with tobacco smoking, that were distinct from cancer sites typically linked to known smoking-related single base substitution (SBS) signatures SBS4 and SBS92 linked to PAHs. These included hematological malignancies and cancers of the kidney, prostate, pancreas and breast, among other sites. In contrast and as expected, tobacco-smoking signatures SBS4 and SBS92 were jointly or individually predominant in cancers of the lung, liver, head-and-neck or bladder. Our study provides first-of-its-kind evidence for the presence of a tobacco-specific nitrosamine mutational signature in human tumors. It suggests that the POB pathway may contribute selectively to the mutational landscapes of certain cancers distinct from those affected by PAH compounds, indicating the potential roles of specific tobacco smoke chemicals in tobacco-linked cancer types lacking SBS4 or SBS92.

RATIONALE:Severe bacterial pneumonia is a leading cause of death worldwide, and mortality rates remain unacceptably high despite appropriate antibiotic treatment. Macrophage membrane-coated nanoparticles (MΦ-NPs) are biomimetic constructs engineered to neutralize bacterial toxins, pathogen-associated molecular patterns, and proinflammatory cytokines. OBJECTIVE:To evaluate the therapeutic potential of MΦ-NPs for the treatment of severe bacterial pneumonia. METHODS:We evaluated the therapeutic potential of MΦ-NPs using in vitro models of infection involving human lung endothelial and epithelial cells, as well as primary human neutrophils, and in vivo murine models of Pseudomonas aeruginosa (PA) and methicillin-resistant Staphylococcus aureus (MRSA) pneumonia. MAIN RESULTS:MΦ-NPs demonstrated potent cytoprotective and anti-inflammatory activity in vitro, without impairing neutrophil antimicrobial function. In both PA and MRSA pneumonia models, MΦ-NP treatment significantly improved survival, reduced bacterial burden, lowered proinflammatory cytokines, and preserved lung architecture. Quantitative proteomics further revealed suppression of inflammatory, coagulation, and fibrotic pathways associated with poor outcomes in human pneumonia and ARDS. CONCLUSIONS:These findings establish MΦ-NPs as a promising host-directed therapeutic strategy for mitigating the deleterious inflammatory sequelae of severe bacterial pneumonia.

Contemporary pathology is increasingly multimodal and requires the application and integration of newer molecular methods to complement classical morphologic evaluation. In diagnostic medical pathology, especially cancer pathology, integrative diagnoses combining morphology, immunohistochemistry, and molecular data including methylation profiling and clinical sequencing are often routine. Such integrative approaches have refined classifications of diseases and enhanced therapeutic decision making. However, in toxicologic pathology, the integration of multiple modalities is relatively nascent. Many pathologists do not have direct and cost-effective access to the information and expertise needed to leverage the availability of advanced molecular modalities. This half-day continuing education course sought to familiarize participants with the evolution of pathology from its early days to state-of-the art applications of molecular, spatial, and computational pathology. The course also aimed to encourage them to implement advanced molecular modalities in their workflows for enhanced understanding of mechanisms of disease and toxicologic processes. This would hopefully facilitate more precise human-relevant translation of preclinical animal model data.

Animal models of infectious pneumonia often require the use of anesthetics, but their choice and impact on outcome is rarely discussed. This study investigates the impact of the most commonly used anesthetics on mortality and bacterial clearance in a murine model of Pseudomonas aeruginosa pneumonia. Isoflurane or ketamine/xylazine were determined to be the most commonly utilized anesthetics for murine pneumonia models. Mice were anesthetized with either ketamine/xylazine or isoflurane during intratracheal infection with P. aeruginosa strains PA14 or PA01. Mortality, bacterial clearance, and lung tissue damage were compared. Additional in vitro assays assessed the effects of ketamine on human whole blood killing, serum killing, and neutrophil functions (reactive oxygen species (ROS) production, neutrophil extracellular trap (NET) production, chemotaxis, and phagocytosis). Mice anesthetized with ketamine/xylazine and infected with PA14 had significantly increased mortality (p = 0.004), and significantly higher bacterial burdens in the blood (p = 0.01) and lungs (p < 0.001). In separate experiments with PA01, mice anesthetized with ketamine/xylazine had significantly increased mortality (p = 0.01), higher bacterial burdens in the blood (p = 0.01), and higher bacterial burdens in the lungs (p = 0.02), along with increased lung tissue pathology (p = 0.03) compared to mice anesthetized with isoflurane. Increased mortality and colony forming units were also observed in mice infected under propofol anesthesia, recovered, and subsequently exposed to ketamine versus control (p = 0.004 and p < 0.001, respectively). Ketamine marginally reduced the killing of PA14 in freshly drawn human whole blood (p = 0.0479), but had no significant effect on the serum’s ability to kill PA14. In addition, ketamine reduced in vitro NETosis and chemotaxis (all p < 0.05), but had no significant effect on ROS production or phagocytosis of human neutrophils. These in vitro effects were observed only at supraclinical ketamine concentrations. Our study emphasizes that the choice of anesthetic impacts key outcomes in murine models of pneumonia, and should therefore be an important consideration in experimental design and when comparing results across different studies.

Urinary neutrophils are a hallmark of urinary tract infection (UTI), yet the mechanisms governing their activation, function, and efficacy in controlling infection remain incompletely understood. Tamm-Horsfall glycoprotein (THP), the most abundant protein in urine, uses terminal sialic acids to bind an inhibitory receptor and dampen neutrophil inflammatory responses. We hypothesized that neutrophil modulation is an integral part of THP-mediated host protection. In a UTI model, THPdeficient mice showed elevated urinary tract bacterial burdens, increased neutrophil recruitment, and more severe tissue histopathological changes compared with WT mice. Furthermore, THPdeficient mice displayed impaired urinary NETosis during UTI. To investigate the effect of THP on NETosis, we coupled in vitro fluorescence-based NET assays, proteomic analyses, and standard and imaging flow cytometry with peripheral human neutrophils. We found that THP increases proteins involved in respiratory chain, neutrophil granules, and chromatin remodeling pathways; enhances NETosis in an ROS-dependent manner; and drives NET-associated morphologic features including nuclear decondensation. These effects were observed only in the presence of a NETosis stimulus and could not be solely replicated with equivalent levels of sialic acid alone. We conclude that THP is a critical regulator of NETosis in the urinary tract, playing a key role in host defense against UTI.

Immune cell infiltration and comparative immune signatures between canine hemangiosarcoma and human angiosarcoma. A, A total of 461 upregulated genes were identified in immune-high (N = 8) compared with immune-low (N = 5) groups in human angiosarcomas (FDR P value < 0.05). B, 567 immune gene signatures were identified among three molecular subtypes of canine hemangiosarcomas (N = 76; FDR P value < 0.001; fold change > 3). The heat maps show upregulated (red) and downregulated (green) genes by unsupervised hierarchical clustering (average linkage; mean-centered; log2 transformed). C, Venn diagram shows 58 common genes associated with signaling pathways of immune cell functions between human and canine tumors. Representative photomicrographs of H&E and IHC staining showing histologic morphology and immune cell infiltration in canine hemangiosarcoma (D) and human angiosarcoma tissues (E) using anti-CD3, anti-PAX5, anti-MAC387, and anti-Iba1 (for canine) or anti-CD163 (for human) antibodies for detecting T cell, B cell, and macrophages. Horseradish peroxidase (counterstain = hematoxylin) or alkaline phosphate (for Iba1; counterstain = methylene blue). Bar = 50 µm. Scatter plots display correlation between transcriptional and IHC immune score in canine hemangiosarcoma (F) and human angiosarcoma (G). Spearman correlation coefficient (R) was calculated.

Establishment of xenografts derived from canine hemangiosarcoma in immunodeficient mice. A, Schematic illustration depicts process of tumor xenografts in BNX mice. B, DHSA-1426 tumor cell line was established from a canine patient diagnosed with hemangiosarcoma (A). Cells from DHSA-1426 passages 5 (p5) and 14 (p14) formed tumors histologically classified as hemangiosarcoma (B and C). Cells cultured from xenograft-derived tumors developed histologically identical tumors after serial transplantation (D). Both the primary tumor (E) and the xenograft tumor (F) were positive for CD31 IHC staining. A–D, Hematoxylin and eosin (H&E) stain. E and F, IHC with an anti-CD31 antibody (alkaline phosphatase conjugates; counterstain = hematoxylin). Bar = 200 µm. C, FISH images using canine-specific (CXCL8, red) and mouse-specific (X chromosome, green) probes in a canine hemangiosarcoma xenograft transplanted into receptive immunodeficient female mouse hosts. Red and green arrows point to representative xenograft canine tumor cells and mouse stromal cells, respectively, to aid in identification. D, Individual points on graph represent relative quantity of donor (dog) and host (mouse) cells in each tumor type. 10–12 fields of pictures at high magnification (400X) per slide were acquired. A total of approximately 1,000 cells in individual xenograft tumor was counted, and the percentages for each species-specific cells are presented. E, Dot plot shows microenvironment scores for 76 primary hemangiosarcoma tissues, estimated using RNA-seq data and xCell algorithm. The red dot indicates a DHSA-1426 tumor tissue used for xenograft.

Hematopoietic expansion derived from adoptive transplantation of canine hemangiosarcoma in immunodeficient mice. A and B, Xenotransplantation of canine hemangiosarcoma cell lines created exuberant myeloid hyperplasia in mouse spleens. Representative photomicrographs show histopathology by H&E staining of spleens from NSG mice transplanted with hemangiosarcoma cell lines SB (A) and EFB (B). C, Immunoreactivity of anti-mouse Ki-67 (Tec-3) antibody shows strongly positive signal in proliferating cells in the spleen. D, Immunostaining of anti-human (and canine cross-reactive) Ki-67 (MiB-1) antibody shows lack of positive staining among proliferating cells. E, The proliferating cells are immunoreactive with anti-mouse Ter-119 antibody. F, SB cells expressing Luciferase are detected in mouse spleen (arrows). Images shown in C, D, E, and F are from IHC staining done in mice inoculated with SB cells modified to express GFP and firefly Luciferase. Horseradish peroxidase (for Ki-67 stains) and alkaline phosphatase (for Ter-119 and Luciferase) conjugates were used. Counterstain = hematoxylin. A–D: Bar = 200 µm; E–F: Bar = 50 µm.

Abstract Hemangiosarcoma and angiosarcoma are soft-tissue sarcomas of blood vessel–forming cells in dogs and humans, respectively. These vasoformative sarcomas are aggressive and highly metastatic, with disorganized, irregular blood-filled vascular spaces. Our objective was to define molecular programs which support the niche that enables progression of canine hemangiosarcoma and human angiosarcoma. Dog-in-mouse hemangiosarcoma xenografts recapitulated the vasoformative and highly angiogenic morphology and molecular characteristics of primary tumors. Blood vessels in the tumors were complex and disorganized, and they were lined by both donor and host cells. In a series of xenografts, we observed that the transplanted hemangiosarcoma cells created exuberant myeloid hyperplasia and gave rise to lymphoproliferative tumors of mouse origin. Our functional analyses indicate that hemangiosarcoma cells generate a microenvironment that supports expansion and differentiation of hematopoietic progenitor populations. Furthermore, gene expression profiling data revealed hemangiosarcoma cells expressed a repertoire of hematopoietic cytokines capable of regulating the surrounding stromal cells. We conclude that canine hemangiosarcomas, and possibly human angiosarcomas, maintain molecular properties that provide hematopoietic support and facilitate stromal reactions, suggesting their potential involvement in promoting the growth of hematopoietic tumors. Significance: We demonstrate that hemangiosarcomas regulate molecular programs supporting hematopoietic expansion and differentiation, providing insights into their potential roles in creating a permissive stromal-immune environment for tumor progression.

Multiple myeloma is a genetically complex and heterogenous malignancy with a 5-year survival rate of approximately 60%. Despite advances in therapy, patients experience cycles of remission and relapse, with each successive line of therapy associated with poorer outcomes; therefore, therapies with different mechanisms of action against new myeloma antigens are needed. G protein–coupled receptor class C group 5 member D (GPRC5D) has emerged as a novel therapeutic target for the treatment of multiple myeloma. We review the biology and target validation of GPRC5D, and clinical data from early phase trials of GPRC5D-targeting bispecific antibodies, talquetamab and forimtamig, and chimeric antigen receptor T cell (CAR-T) therapies, MCARH109, OriCAR-017, and BMS-986393. In addition to adverse events (AEs) associated with T-cell–redirection therapies irrespective of target, a consistent pattern of dermatologic and oral AEs has been reported across several trials of GPRC5D-targeting bispecific antibodies, as well as rare cerebellar events with CAR-T therapy. Additional studies are needed to understand the underlying mechanisms involved in the development of skin- and oral-related toxicities. We review the strategies that have been used to manage these GPRC5D-related toxicities. Preliminary efficacy data showed overall response rates for GPRC5D-targeting T-cell–redirecting therapies were ≥64%; most responders achieved a very good partial response or better. Pharmacokinetics/pharmacodynamics showed that these therapies led to cytokine release and T-cell activation. In conclusion, results from early phase trials of GPRC5D-targeting T-cell–redirecting agents have shown promising efficacy and manageable safety profiles, including lower infection rates compared with B-cell maturation antigen- and Fc receptor-like protein 5-targeting bispecific antibodies. Further clinical trials, including those investigating GPRC5D-targeting T-cell–redirecting agents in combination with other anti-myeloma therapies and with different treatment modalities, may help to elucidate the future optimal treatment regimen and sequence for patients with multiple myeloma and improve survival outcomes.

Hematopoietic support and stromal regulation of canine hemangiosarcoma cells. A, Flow cytometric data depict populations of cells expressing CD43 and CD45 differentiated from CD34+ hUCB cells. CD34+ hUCB cells were pooled from 2 patients. M2-10B4, hBM-MSCs, and canine hemangiosarcoma cells (DHSA-1426 and EFB) were seeded on gelatin-coated 24-well plates at a density of 1 × 105 cells/well. Gelatin-coated wells without stroma served as a negative control. Surface antigens CD34, CD43, and CD45 were analyzed at week 5. B and C, Bar graphs illustrate number of different colonies formed by hUCB CD34+ cells cocultured with feeder cells. Both DHSA-1426 and EFB canine hemangiosarcoma cell lines expanded hUCB CD34+ cells similarly to the M2-10B4 and hBM-MSC positive control lines, while gelatin-coated wells alone failed to support expansion. Burst-forming unit-erythroid (BFU-E), CFU (colony-forming unit)-Erythroid (CFU-E), CFU-granulocyte/macrophage (CFU-GM), CFU-macrophage (CFU-M), and CFU-granulocyte/erythroid/macrophage/megakaryocyte (CFU-GEMM) were determined for CFU assay.

Tobacco usage is linked to multiple cancer types and accounts for a quarter of all cancer-related deaths. Tobacco smoke contains various carcinogenic compounds, including polycyclic aromatic hydrocarbons (PAH), though the mutagenic potential of many tobacco-related chemicals remains largely unexplored. In particular, the highly carcinogenic tobacco-specific nitrosamines NNN and NNK form pre-mutagenic pyridyloxobutyl (POB) DNA adducts. In the study presented here, we identified genome-scale POB-induced mutational signatures in cell lines and rat tumors, while also investigating their role in human cancer. These signatures are characterized by T>N and C>T mutations forming from specific POB adducts damaging dT and dC residues. Analysis of 2,780 cancer genomes uncovered POB signatures in ∼180 tumors; from cancer types distinct from the ones linked to smoking-related signatures SBS4 and SBS92. This suggests that, unlike PAH compounds, the POB pathway may contribute uniquely to the mutational landscapes of certain hematological malignancies and cancers of the kidney, breast, prostate and pancreas.

Gene expression analysis in DHSA-1426 hemangiosarcoma cells and xenograft tumors using RNA-seq data. A, The volcano plot visualizes 2,391 significant DEGs (1,034 upregulated and 1,357 downregulated) in DHSA-1426 cells (n = 3) compared with nonmalignant endothelial cells (n = 4; q-value < 0.01; log2 fold change > |2|). The DEGs are indicated by red dots. NS = not significant; log2FC = log2 fold change. B, Gene Ontology enrichment analysis depicts biological processes associated with significant DEGs. P, adjust = adjusted P-value. C, Bar graphs present normalized gene expression values of CSF3, IL6, and IL8 in RNA-seq data generated from canine nonmalignant endothelial cells (n = 4) and DHSA-1426 hemangiosarcoma cells (n = 3). D, Schematic illustration visualizes the experimental steps for species-specific gene expression analysis of xenograft tumors using RNA-seq data. E, Bar graphs show the expression of mouse-specific genes in RNA-seq data generated from xenograft tumor tissues of canine hemangiosarcoma (n = 4) and mouse lymphoma (n = 7). A two-way ANOVA test was conducted to compare the means between groups. ****, P < 0.0001