Acute intestinal inflammation involves early accumulation of neutrophils (PMNs) followed by either resolution or progression to chronic inflammation. Based on recent evidence that mucosal metabolism ...influences disease outcomes, we hypothesized that transmigrating PMNs influence the transcriptional profile of the surrounding mucosa. Microarray studies revealed a cohort of hypoxia-responsive genes regulated by PMN-epithelial crosstalk. Transmigrating PMNs rapidly depleted microenvironmental O2 sufficiently to stabilize intestinal epithelial cell hypoxia-inducible factor (HIF). By utilizing HIF reporter mice in an acute colitis model, we investigated the relative contribution of PMNs and the respiratory burst to “inflammatory hypoxia” in vivo. CGD mice, lacking a respiratory burst, developed accentuated colitis compared to control, with exaggerated PMN infiltration and diminished inflammatory hypoxia. Finally, pharmacological HIF stabilization within the mucosa protected CGD mice from severe colitis. In conclusion, transcriptional imprinting by infiltrating neutrophils modulates the host response to inflammation, via localized O2 depletion, resulting in microenvironmental hypoxia and effective inflammatory resolution.
Display omitted
•Infiltrating PMNs consume sufficient O2 to render adjacent colonic epithelia hypoxic•Hypoxia reporter mice demonstrate PMN-dependent hypoxia in inflamed colitic lesions•CGD mice do not incite mucosal hypoxia and develop severe nonresolving colitis•Mucosal HIF stabilization ameliorates colitis severity in wild-type and CGD mice
The complement cascade is a part of the innate immune system that acts primarily to remove pathogens and injured cells. However, complement activation is also peculiarly associated with tumor ...progression. Here we report mechanistic insights into this association in multiple immunocompetent orthotopic models of lung cancer. After tumor engraftment, we observed systemic activation of the complement cascade as reflected by elevated levels of the key regulator C3a. Notably, growth of primary tumors and metastases was both strongly inhibited in C3-deficient mice (C3
mice), with tumors undetectable in many subjects. Growth inhibition was associated with increased numbers of IFNγ
/TNFα
/IL10
CD4
and CD8
T cells. Immunodepletion of CD4
but not CD8
T cells in tumor-bearing subjects reversed the inhibitory effects of C3 deletion. Similarly, antagonists of the C3a or C5a receptors inhibited tumor growth. Investigations using multiple tumor cell lines in the orthotopic model suggested the involvement of a C3/C3 receptor autocrine signaling loop in regulating tumor growth. Overall, our findings offer functional evidence that complement activation serves as a critical immunomodulator in lung cancer progression, acting to drive immune escape via a C3/C5-dependent pathway.
This provocative study suggests that inhibiting complement activation may heighten immunotherapeutic responses in lung cancer, offering findings with immediate implications, given the existing clinical availability of complement antagonists.
.
Macrophages represent an important component of the tumor microenvironment and play a complex role in cancer progression. These cells are characterized by a high degree of plasticity, and they alter ...their phenotype in response to local environmental cues. Whereas the M1/M2 classification of macrophages has been widely used, the complexity of macrophage phenotypes has not been well studied, particularly in lung cancer. In this study we employed an orthotopic immunocompetent model of lung adenocarcinoma in which murine lung cancer cells are directly implanted into the left lobe of syngeneic mice. Using multimarker flow cytometry, we defined and recovered several distinct populations of monocytes/macrophages from tumors at different stages of progression. We used RNA-seq transcriptional profiling to define distinct features of each population and determine how they change during tumor progression. We defined an alveolar resident macrophage population that does not change in number and expresses multiple genes related to lipid metabolism and lipid signaling. We also defined a population of tumor-associated macrophages that increase dramatically with tumor and selectively expresses a panel of chemokine genes. A third population, which resembles tumor-associated monocytes, expresses a large number of genes involved in matrix remodeling. By correlating transcriptional profiles with clinically prognostic genes, we show that specific monocyte/macrophage populations are enriched in genes that predict outcomes in lung adenocarcinoma, implicating these subpopulations as critical determinants of patient survival. Our data underscore the complexity of monocytes/macrophages in the tumor microenvironment, and they suggest that distinct populations play specific roles in tumor progression.
Epidermal growth factor receptor (EGFR) is frequently amplified or overexpressed in head and neck squamous cell carcinoma (HNSCC) and is a clinically validated target for the therapeutic antibody, ...cetuximab, in the management of this cancer. The degree of response to EGFR inhibitors measured by tumor shrinkage varies widely among HNSCC patients, and the biological mechanisms that underlie therapeutic heterogeneity amongst HNSCC patients remain ill-defined.
EGFR-dependent human and murine HNSCC cell lines were treated with the EGFR/ERBB inhibitors, gefitinib and AZD8931, and submitted to RNAseq, GSEA, and qRT-PCR. Conditioned media was analyzed by ELISA and Luminex assays. Murine HNSCC tumors were stained for T cell markers by immunofluorescence. Primary HSNCC patient specimens treated with single agent cetuximab were stained with Vectra multispectral immunofluorescence.
The transcriptional reprogramming response to EGFR/ERBB-specific TKIs was measured in a panel of EGFR-dependent human HNSCC cell lines and interferon (IFN) α and γ responses identified as top-ranked TKI-induced pathways. Despite similar drug sensitivity, responses among 7 cell lines varied quantitatively and qualitatively, especially regarding the induced chemokine and cytokine profiles. Of note, the anti-tumorigenic chemokine, CXCL10, and the pro-tumorigenic factor, IL6, exhibited wide-ranging and non-overlapping induction. Similarly, AZD8931 exerted potent growth inhibition, IFNα/IFNγ pathway activation, and CXCL10 induction in murine B4B8 HNSCC cells. AZD8931 treatment of immune-competent mice bearing orthotopic B4B8 tumors increased CD8 + T cell content and the therapeutic response was abrogated in nu/nu mice relative to BALB/c mice. Finally, Vectra 3.0 analysis of HNSCC patient tumors prior to and after 3-4 weeks of single agent cetuximab treatment revealed increased CD8 + T cell content in specimens from patients exhibiting a therapeutic response relative to non-responders.
The findings reveal heterogeneous, tumor cell-intrinsic, EGFR/ERBB inhibitor-induced IFN pathway activation in HNSCC and suggest that individual tumor responses to oncogene-targeted agents are a sum of direct growth inhibitory effects and variably-induced participation of host immune cells.
Abstract
Cardiovascular diseases are characterized by chronic vascular dysfunction and provoke pathological remodelling events, such as neointima formation, atherosclerotic lesion development, and ...adventitial fibrosis. While lineage-tracing studies have shown that phenotypically modulated smooth muscle cells (SMCs) are the major cellular component of neointimal lesions, the cellular origins and microenvironmental signalling mechanisms that underlie remodelling along the adventitial vascular layer are not fully understood. However, a growing body of evidence supports a unique population of adventitial lineage-restricted progenitor cells expressing the stem cell marker, stem cell antigen-1 (Sca1; AdvSca1 cells) as important effectors of adventitial remodelling and suggests that they are at least partially responsible for subsequent pathological changes that occur in the media and intima. AdvSca1 cells are being studied in murine models of atherosclerosis, perivascular fibrosis, and neointima formation in response to acute vascular injury. Depending on the experimental conditions, AdvSca1 cells exhibit the capacity to differentiate into SMCs, endothelial cells, chondrocytes, adipocytes, and pro-remodelling cells, such as myofibroblasts and macrophages. These data indicate that AdvSca1 cells may be a targetable cell population to influence the outcomes of pathologic vascular remodelling. Important questions remain regarding the origins of AdvSca1 cells and the essential signalling mechanisms and microenvironmental factors that regulate both maintenance of their stem-like, progenitor phenotype and their differentiation into lineage-specified cell types. Adding complexity to the story, recent data indicate that the collective population of adventitial progenitor cells is likely composed of several smaller, lineage-restricted subpopulations, which are not fully defined by their transcriptomic profile and differentiation capabilities. The aim of this review is to outline the heterogeneity of Sca1+ adventitial progenitor cells, summarize their role in vascular homeostasis and remodelling, and comment on their translational relevance in humans.
Graphical Abstract
Activation of peroxisome proliferator-activated receptor-γ (PPARγ) inhibits growth of cancer cells including non-small cell lung cancer (NSCLC). Clinically, use of thiazolidinediones, which are ...pharmacological activators of PPARγ is associated with a lower risk of developing lung cancer. However, the role of this pathway in lung cancer metastasis has not been examined well. The systemic effect of pioglitazone was examined in two models of lung cancer metastasis in immune-competent mice. In an orthotopic model, murine lung cancer cells implanted into the lungs of syngeneic mice metastasized to the liver and brain. As a second model, cancer cells injected subcutaneously metastasized to the lung. In both models systemic administration of pioglitazone increased the rate of metastasis. Examination of tissues from the orthotopic model demonstrated increased numbers of arginase I-positive macrophages in tumors from pioglitazone-treated animals. In co-culture experiments of cancer cells with bone marrow-derived macrophages, pioglitazone promoted arginase I expression in macrophages and this was dependent on the expression of PPARγ in the macrophages. To assess the contribution of PPARγ in macrophages to cancer progression, experiments were performed in bone marrow-transplanted animals receiving bone marrow from Lys-M-Cre+/PPARγ(flox/flox) mice, in which PPARγ is deleted specifically in myeloid cells (PPARγ-Mac(neg)), or control PPARγ(flox/flox) mice. In both models, mice receiving PPARγ-Mac(neg) bone marrow had a marked decrease in secondary tumors which was not significantly altered by treatment with pioglitazone. This was associated with decreased numbers of arginase I-positive cells in the lung. These data support a model in which activation of PPARγ may have opposing effects on tumor progression, with anti-tumorigenic effects on cancer cells, but pro-tumorigenic effects on cells of the microenvironment, specifically myeloid cells.
Vascular disease progression is associated with marked changes in vascular smooth muscle cell (SMC) phenotype and function. SMC contractile gene expression and, thus differentiation, is under direct ...transcriptional control by the transcription factor, serum response factor (SRF); however, the mechanisms dynamically regulating SMC phenotype are not fully defined. Here we report that the lipid and protein phosphatase, PTEN, has a novel role in the nucleus by functioning as an indispensible regulator with SRF to maintain the differentiated SM phenotype. PTEN interacts with the N-terminal domain of SRF and PTEN-SRF interaction promotes SRF binding to essential promoter elements in SM-specific genes. Factors inducing phenotypic switching promote loss of nuclear PTEN through nucleo-cytoplasmic translocation resulting in reduced myogenically active SRF, but enhanced SRF activity on target genes involved in proliferation. Overall decreased expression of PTEN was observed in intimal SMCs of human atherosclerotic lesions underlying the potential clinical importance of these findings.
Despite widespread expression of epidermal growth factor (EGF) receptors (EGFRs) and EGF family ligands in non-small-cell lung cancer (NSCLC), EGFR-specific tyrosine kinase inhibitors (TKIs) such as ...gefitinib exhibit limited activity in this cancer. We propose that autocrine growth signaling pathways distinct from EGFR are active in NSCLC cells. To this end, gene expression profiling revealed frequent coexpression of specific fibroblast growth factors (FGFs) and FGF receptors (FGFRs) in NSCLC cell lines. It is noteworthy that FGF2 and FGF9 as well as FGFR1 IIIc and/or FGFR2 IIIc mRNA and protein are frequently coexpressed in NSCLC cell lines, especially those that are insensitive to gefitinib. Specific silencing of FGF2 reduced anchorage-independent growth of two independent NSCLC cell lines that secrete FGF2 and coexpress FGFR1 IIIc and/or FGFR2 IIIc. Moreover, a TKI (+/-)-1-(anti-3-hydroxy-cyclopentyl)-3-(4-methoxy-phenyl)-7-phenylamino-3,4-dihydro-1H-pyrimido-4,5-dpyrimidin-2-one (RO4383596) that targets FGFRs inhibited basal FRS2 and extracellular signal-regulated kinase phosphorylation, two measures of FGFR activity, as well as proliferation and anchorage-independent growth of NSCLC cell lines that coexpress FGF2 or FGF9 and FGFRs. By contrast, RO4383596 influenced neither signal transduction nor growth of NSCLC cell lines lacking FGF2, FGF9, FGFR1, or FGFR2 expression. Thus, FGF2, FGF9 and their respective high-affinity FGFRs comprise a growth factor autocrine loop that is active in a subset of gefitinib-insensitive NSCLC cell lines.
Immunotherapy clinical trials targeting the programmed-death ligand axis (PD-1/PD-L1) show that most head and neck squamous cell carcinoma (HNSCC) patients are resistant to PD-1/PD-L1 inhibition. We ...investigated whether local radiation to the tumor can transform the immune landscape and render poorly immunogenic HNSCC tumors sensitive to PD-L1 inhibition. We used the first novel orthotopic model of HNSCC with genetically distinct murine cell lines. Tumors were resistant to PD-L1 checkpoint blockade, harbored minimal PD-L1 expression and tumor infiltrating lymphocytes at baseline, and were resistant to radiotherapy. The combination of radiation and PD-L1 inhibition significantly enhanced tumor control and improved survival. This was mediated in part through upregulation of PD-L1 on tumor cells and increased T-cell infiltration after RT, resulting in a highly inflamed tumor. Depletion of both CD4 and CD8 T-cells completely abrogated the effect of anti PD-L1 with radiation on tumor growth. Our findings provide evidence that radiation to the tumor can induce sensitivity to PD-L1 checkpoint blockade in orthotopic models of HNSCC. These findings have direct relevance to high risk HNSCC patients with poorly immunogenic tumors and who may benefit from combined radiation and checkpoint blockade.
In recent years, fate-mapping lineage studies in mouse models have led to major advances in vascular biology by allowing investigators to track specific cell populations in vivo. One of the most ...frequently used lineage tracing approaches involves tamoxifen-inducible Cre
-LoxP systems. However, tamoxifen treatment can also promote effects independent of Cre recombinase activation, many of which have not been fully explored.
To elucidate off-target effects of tamoxifen, male and female mice were either unmanipulated or injected with tamoxifen or corn oil. All mice received PCSK9 (proprotein convertase subtilisin/kexin type 9)-AAV (adeno-associated virus) injections and a modified Western diet to induce hypercholesterolemia. After 2 weeks, serum cholesterol and liver morphology were assessed. To determine the duration of any tamoxifen effects in long-term atherosclerosis experiments, mice received either 12 days of tamoxifen at baseline or 12 days plus 2 sets of 5-day tamoxifen boosters; all mice received PCSK9-AAV injections and a modified Western diet to induce hypercholesterolemia. After 24 weeks, serum cholesterol and aortic sinus plaque burden were measured.
After 2 weeks of atherogenic treatment, mice injected with tamoxifen demonstrated significantly reduced serum cholesterol levels compared with uninjected- or corn oil-treated mice. However, there were no differences in PCSK9-mediated knockdown of LDL (low-density lipoprotein) receptors between the groups. Additionally, tamoxifen-treated mice exhibited significantly increased hepatic lipid accumulation compared with the other groups. Finally, the effects of tamoxifen remained for at least 8 weeks after completion of injections, with mice demonstrating persistent decreased serum cholesterol and impaired atherosclerotic plaque formation.
In this study, we establish that tamoxifen administration results in decreased serum cholesterol, decreased plaque formation, and increased hepatic lipid accumulation. These alterations represent significant confounding variables in atherosclerosis research, and we urge future investigators to take these findings into consideration when planning and executing their own atherosclerosis experiments.
PDF
