Myofibroblastic cancer-associated fibroblast subtype heterogeneity in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has a fibrotic stroma that has both tumor-promoting and tumor-restraining properties. Different types of cancer-associated fibroblasts (CAFs) have been described. Here, we investigated whether CAFs within the same subtype exhibit heterogeneous functions. METHODS: We evaluated the gene and protein expression differences in two myofibroblastic CAF (myCAF) lines using single-cell and bulk RNA-sequencing. We utilized proliferation and migration assays to determine the effect of different CAF lines on a tumor cell line. RESULTS: We found that myCAF lines express an activated stroma subtype gene signature, which is associated with a shorter survival in patients. Although both myCAF lines expressed α-smooth muscle actin (α-SMA), platelet-derived growth factor-α (PDGFR-α), fibroblast-activated protein (FAP), and vimentin, we observed heterogeneity between the two lines. Similarly, despite being consistent with myCAF gene expression overall, heterogeneity within specific genes was observed. We found that these differences extended to the functional level where the two myCAF lines had different effects on the same tumor cell line. The myCAF216 line, which had slightly increased inflammatory CAF-like gene expression and higher protein expression of α-SMA, PDGFR-α, and FAP was found to restrain migration of tumor cells. CONCLUSIONS: We found that two myCAF lines with globally similar expression characteristics had different effects on the same tumor cell line, one promoting and the other restraining migration. Our study highlights that there may be unappreciated heterogeneity within CAF subtypes. Further investigation and attention to specific genes or proteins that may drive this heterogeneity will be important.

Novel leptin receptor signaling mutants identify location and sex-dependent modulation of bone density, adiposity, and growth.

Leptin, a hormone primarily produced by adipocytes, contributes to the regulation of bone health by modulating bone density, growth and adiposity. Upon leptin binding, multiple sites of the long form of the leptin receptor (LepRb) are phosphorylated to trigger activation of downstream signaling pathways. To address the role of LepRb-signaling pathways in bone health, we compared the effects of three LepRb mutations on bone density, adiposity, and growth in male and female mice. The ∆65 mutation, which lacks the known tyrosine phosphorylation sites, caused obesity and the most dramatic bone phenotype marked by excessive bone adiposity, osteoporosis, and decreased growth, consistent with the phenotype of db/db and ob/ob mice that fully lack leptin receptor signaling. Mutation of LepRb Tyr 1138 , which results in an inability to recruit and phosphorylate signal transducer and activator of transcription 3, also caused obesity, but bone loss and adiposity were more dominant in male mice and no growth defect was observed. In contrast, mutation of LepRb Tyr 985 , which blocks SHP2/SOCS3 recruitment to LepRb and contributes to leptin hypersensitivity, promoted increased femur bone density only in male mice, while marrow adiposity and bone growth were not affected. Additional analyses of vertebral trabecular bone volume indicate that only the Tyr 1138 mutant mice exhibit bone loss in vertebrae. Together, our findings suggest that the phosphorylation status of specific sites of the LepRb contribute to the sex- and location-dependent bone responses to leptin. Unraveling the mechanisms by which leptin responses are sex- and location-dependent can contribute to the development of uniquely targeted osteoporosis therapies.

Bacterial Dose-Dependent Role of G Protein-Coupled Receptor Kinase 5 in Escherichia coli-Induced Pneumonia.

G protein-coupled receptor kinase 5 (GRK5) is a serine/threonine kinase previously shown to mediate polymicrobial sepsis-induced inflammation. The goal of the present study was to examine the role of GRK5 in monomicrobial pulmonary infection by using an intratracheal Escherichia coli infection model of pneumonia. We used sublethal and lethal doses of E. coli to examine the mechanistic differences between low-grade and high-grade inflammation induced by E. coli infection. With a sublethal dose of E. coli, GRK5 knockout (KO) mice exhibited higher plasma CXCL1/KC levels and enhanced lung neutrophil recruitment early after infection, and lower bacterial loads, than wild-type (WT) mice. The inflammatory response was also diminished, and resolution of inflammation advanced, in the lungs of GRK5 KO mice. In contrast to the reduced bacterial loads in GRK5 KO mice following a sublethal dose, at a lethal dose of E. coli, the bacterial burdens remained high in GRK5 KO mice relative to those in WT mice. This occurred in spite of enhanced plasma CXCL1 levels as well as neutrophil recruitment in the KO mice. But the recruited neutrophils (following high-dose infection) exhibited decreased CD11b expression and reduced reactive oxygen species production, suggesting decreased neutrophil activation or increased neutrophil exhaustion in the GRK5 KO mice. In agreement with the increased bacterial burden, KO mice showed poorer survival than WT mice following E. coli infection at a lethal dose. Overall, our data suggest that GRK5 negatively regulates CXCL1/KC levels during bacterial pneumonia but that the role of GRK5 in the clinical outcome in this model is dependent on the bacterial dose.

Determination of permissive and restraining cancer-associated fibroblast (DeCAF) subtypes.

Cancer-associated fibroblast (CAF) subpopulations in pancreatic ductal adenocarcinoma (PDAC) have been identified using single-cell RNA sequencing (scRNAseq) with divergent characteristics, but their clinical relevance remains unclear. We translate scRNAseq-derived CAF cell-subpopulation-specific marker genes to bulk RNAseq data, and develop a single- sample classifier, DeCAF, for the classification of clinically rest raining and perm issive CAF subtypes. We validate DeCAF in 19 independent bulk transcriptomic datasets across four tumor types (PDAC, mesothelioma, bladder and renal cell carcinoma). DeCAF subtypes have distinct histology features, immune landscapes, and are prognostic and predict response to therapy across cancer types. We demonstrate that DeCAF is clinically replicable and robust for the classification of CAF subtypes in patients for multiple tumor types, providing a better framework for the future development and translation of therapies against permissive CAF subtypes and preservation of restraining CAF subtypes. Significance: We introduce a replicable and robust classifier, DeCAF, that delineates the significance of the role of permissive and restraining CAF subtypes in cancer patients. DeCAF is clinically tractable, prognostic and predictive of treatment response in multiple cancer types and lays the translational groundwork for the preclinical and clinical development of CAF subtype specific therapies.