Identification of the Human Skeletal Stem Cell.

Stem cell regulation and hierarchical organization of human skeletal progenitors remain largely unexplored. Here, we report the isolation of a self-renewing and multipotent human skeletal stem cell (hSSC) that generates progenitors of bone, cartilage, and stroma, but not fat. Self-renewing and multipotent hSSCs are present in fetal and adult bones and can also be derived from BMP2-treated human adipose stroma (B-HAS) and induced pluripotent stem cells (iPSCs). Gene expression analysis of individual hSSCs reveals overall similarity between hSSCs obtained from different sources and partially explains skewed differentiation toward cartilage in fetal and iPSC-derived hSSCs. hSSCs undergo local expansion in response to acute skeletal injury. In addition, hSSC-derived stroma can maintain human hematopoietic stem cells (hHSCs) in serum-free culture conditions. Finally, we combine gene expression and epigenetic data of mouse skeletal stem cells (mSSCs) and hSSCs to identify evolutionarily conserved and divergent pathways driving SSC-mediated skeletogenesis. VIDEO ABSTRACT.

Identification and specification of the mouse skeletal stem cell.

How are skeletal tissues derived from skeletal stem cells? Here, we map bone, cartilage, and stromal development from a population of highly pure, postnatal skeletal stem cells (mouse skeletal stem cells, mSSCs) to their downstream progenitors of bone, cartilage, and stromal tissue. We then investigated the transcriptome of the stem/progenitor cells for unique gene-expression patterns that would indicate potential regulators of mSSC lineage commitment. We demonstrate that mSSC niche factors can be potent inducers of osteogenesis, and several specific combinations of recombinant mSSC niche factors can activate mSSC genetic programs in situ, even in nonskeletal tissues, resulting in de novo formation of cartilage or bone and bone marrow stroma. Inducing mSSC formation with soluble factors and subsequently regulating the mSSC niche to specify its differentiation toward bone, cartilage, or stromal cells could represent a paradigm shift in the therapeutic regeneration of skeletal tissues.

Aged skeletal stem cells generate an inflammatory degenerative niche.

Loss of skeletal integrity during ageing and disease is associated with an imbalance in the opposing actions of osteoblasts and osteoclasts1. Here we show that intrinsic ageing of skeletal stem cells (SSCs)2 in mice alters signalling in the bone marrow niche and skews the differentiation of bone and blood lineages, leading to fragile bones that regenerate poorly. Functionally, aged SSCs have a decreased bone- and cartilage-forming potential but produce more stromal lineages that express high levels of pro-inflammatory and pro-resorptive cytokines. Single-cell RNA-sequencing studies link the functional loss to a diminished transcriptomic diversity of SSCs in aged mice, which thereby contributes to the transformation of the bone marrow niche. Exposure to a youthful circulation through heterochronic parabiosis or systemic reconstitution with young haematopoietic stem cells did not reverse the diminished osteochondrogenic activity of aged SSCs, or improve bone mass or skeletal healing parameters in aged mice. Conversely, the aged SSC lineage promoted osteoclastic activity and myeloid skewing by haematopoietic stem and progenitor cells, suggesting that the ageing of SSCs is a driver of haematopoietic ageing. Deficient bone regeneration in aged mice could only be returned to youthful levels by applying a combinatorial treatment of BMP2 and a CSF1 antagonist locally to fractures, which reactivated aged SSCs and simultaneously ablated the inflammatory, pro-osteoclastic milieu. Our findings provide mechanistic insights into the complex, multifactorial mechanisms that underlie skeletal ageing and offer prospects for rejuvenating the aged skeletal system.

Identification of a novel gene signature for neuroblastoma differentiation using a Boolean implication network.

Although induction of differentiation represents an effective strategy for neuroblastoma treatment, the mechanisms underlying neuroblastoma differentiation are poorly understood. We generated a computational model of neuroblastoma differentiation consisting of interconnected gene clusters identified based on symmetric and asymmetric gene expression relationships. We identified a differentiation signature consisting of series of gene clusters comprised of 1251 independent genes that predicted neuroblastoma differentiation in independent datasets and in neuroblastoma cell lines treated with agents known to induce differentiation. This differentiation signature was associated with patient outcomes in multiple independent patient cohorts and validated the role of MYCN expression as a marker of neuroblastoma differentiation. Our results further identified novel genes associated with MYCN via asymmetric Boolean implication relationships that would not have been identified using symmetric computational approaches and that were associated with both neuroblastoma differentiation and patient outcomes. Our differentiation signature included a cluster of genes involved in intracellular signaling and growth factor receptor trafficking pathways that is strongly associated with neuroblastoma differentiation, and we validated the associations of UBE4B, a gene within this cluster, with neuroblastoma cell and tumor differentiation. Our findings demonstrate that Boolean network analyses of symmetric and asymmetric gene expression relationships can identify novel genes and pathways relevant for neuroblastoma tumor differentiation that could represent potential therapeutic targets.

Artificial intelligence-guided discovery of gastric cancer continuum.

BACKGROUND: Detailed understanding of pre-, early and late neoplastic states in gastric cancer helps develop better models of risk of progression to gastric cancers (GCs) and medical treatment to intercept such progression. METHODS: We built a Boolean implication network of gastric cancer and deployed machine learning algorithms to develop predictive models of known pre-neoplastic states, e.g., atrophic gastritis, intestinal metaplasia (IM) and low- to high-grade intestinal neoplasia (L/HGIN), and GC. Our approach exploits the presence of asymmetric Boolean implication relationships that are likely to be invariant across almost all gastric cancer datasets. Invariant asymmetric Boolean implication relationships can decipher fundamental time-series underlying the biological data. Pursuing this method, we developed a healthy mucosa → GC continuum model based on this approach. RESULTS: Our model performed better against publicly available models for distinguishing healthy versus GC samples. Although not trained on IM and L/HGIN datasets, the model could identify the risk of progression to GC via the metaplasia → dysplasia → neoplasia cascade in patient samples. The model could rank all publicly available mouse models for their ability to best recapitulate the gene expression patterns during human GC initiation and progression. CONCLUSIONS: A Boolean implication network enabled the identification of hitherto undefined continuum states during GC initiation. The developed model could now serve as a starting point for rationalizing candidate therapeutic targets to intercept GC progression.

Boolean implication analysis of single-cell data predicts retinal cell type markers.

BACKGROUND: The retina is a complex tissue containing multiple cell types that are essential for vision. Understanding the gene expression patterns of various retinal cell types has potential applications in regenerative medicine. Retinal organoids (optic vesicles) derived from pluripotent stem cells have begun to yield insights into the transcriptomics of developing retinal cell types in humans through single cell RNA-sequencing studies. Previous methods of gene reporting have relied upon techniques in vivo using microarray data, or correlational and dimension reduction methods for analyzing single cell RNA-sequencing data computationally. We aimed to develop a state-of-the-art Boolean method that filtered out noise, could be applied to a wide variety of datasets and lent insight into gene expression over differentiation. RESULTS: Here, we present a bioinformatic approach using Boolean implication to discover genes which are retinal cell type-specific or involved in retinal cell fate. We apply this approach to previously published retina and retinal organoid datasets and improve upon previously published correlational methods. Our method improves the prediction accuracy of marker genes of retinal cell types and discovers several new high confidence cone and rod-specific genes. CONCLUSIONS: The results of this study demonstrate the benefits of a Boolean approach that considers asymmetric relationships. We have shown a statistically significant improvement from correlational, symmetric methods in the prediction accuracy of retinal cell-type specific genes. Furthermore, our method contains no cell or tissue-specific tuning and hence could impact other areas of gene expression analyses in cancer and other human diseases.

A long isoform of GIV/Girdin contains a PDZ-binding module that regulates localization and G-protein binding.

PDZ domains are one of the most abundant protein domains in eukaryotes and are frequently found on junction-localized scaffold proteins. Various signaling molecules bind to PDZ proteins via PDZ-binding motifs (PBM) and fine-tune cellular signaling. However, how such interaction affects protein function is difficult to predict and must be solved empirically. Here we describe a long isoform of the guanine nucleotide exchange factor GIV/Girdin (CCDC88A) that we named GIV-L, which is conserved throughout evolution, from invertebrates to vertebrates, and contains a PBM. Unlike GIV, which lacks PBM and is cytosolic, GIV-L localizes onto cell junctions and has a PDZ interactome (as shown through annotating Human Cell Map and BioID-proximity labeling studies), which impacts GIV-L's ability to bind and activate trimeric G-protein, Gαi, through its guanine-nucleotide exchange modulator (GEM) module. This GEM module is found exclusively in vertebrates. We propose that the two functional modules in GIV may have evolved sequentially: the ability to bind PDZ proteins via the PBM evolved earlier in invertebrates, whereas G-protein binding and activation may have evolved later only among vertebrates. Phenotypic studies in Caco-2 cells revealed that GIV and GIV-L may have antagonistic effects on cell growth, proliferation (cell cycle), and survival. Immunohistochemical analysis in human colon tissues showed that GIV expression increases with a concomitant decrease in GIV-L during cancer initiation. Taken together, these findings reveal how regulation in GIV/CCDC88A transcript helps to achieve protein modularity, which allows the protein to play opposing roles either as a tumor suppressor (GIV-L) or as an oncogene (GIV).

Bladder Cancer Invasion Is Mediated by Mammalian Target of Rapamycin Complex 2-Driven Regulation of Nitric Oxide and Invadopodia Formation.

Bladder cancer invasion depends on mammalian target of rapamycin complex 2 (mTORC2) activity, although the downstream mTORC2 effectors that mediate this effect have not been fully defined. One potential downstream effector is the arginine derivative nitric oxide (NO). This study identified a stage-associated increase in the expression of the NO-generating enzymes endothelial NO synthase (eNOS) and inducible NOS (iNOS) in human bladder cancer. Reduction of NOS activity by pharmacologic inhibition or silencing of NOS enzymes reduced cancer cell invasion, with similar effects observed using the NO scavenger cobinamide. By contrast, enhanced invasion was seen with the NO donor Deta-NONOate and an analog of the downstream NO second messenger cGMP. Next, NOS expression was evaluated in invadopodia, which are cellular protrusions that form the invasive tips of cancer cells. Invadopodia were enriched in both iNOS protein and mTORC2 activity, and invadopodia formation was increased by Deta-NONOate and decreased by cobinamide and ablation of mTORC2 activity. Additionally, mTORC2 increased expression of iNOS. Using a zebrafish model, injection of iNOS- or rictor-silenced cells reduced the frequency of bladder cancer cell metastasis in zebrafish. These results indicate that mTORC2 can mediate bladder cancer cell invasion through increased iNOS expression, resulting in increased NO and cGMP production in invadopodia and further propagation of invadopodia formation.

Hoxb5 marks long-term haematopoietic stem cells and reveals a homogenous perivascular niche.

Haematopoietic stem cells (HSCs) are arguably the most extensively characterized tissue stem cells. Since the identification of HSCs by prospective isolation, complex multi-parameter flow cytometric isolation of phenotypic subsets has facilitated studies on many aspects of HSC biology, including self-renewal, differentiation, ageing, niche, and diversity. Here we demonstrate by unbiased multi-step screening, identification of a single gene, homeobox B5 (Hoxb5, also known as Hox-2.1), with expression in the bone marrow that is limited to long-term (LT)-HSCs in mice. Using a mouse single-colour tri-mCherry reporter driven by endogenous Hoxb5 regulation, we show that only the Hoxb5(+) HSCs exhibit long-term reconstitution capacity after transplantation in primary transplant recipients and, notably, in secondary recipients. Only 7-35% of various previously defined immunophenotypic HSCs are LT-HSCs. Finally, by in situ imaging of mouse bone marrow, we show that >94% of LT-HSCs (Hoxb5(+)) are directly attached to VE-cadherin(+) cells, implicating the perivascular space as a near-homogenous location of LT-HSCs.

Boolean implication analysis unveils candidate universal relationships in microbiome data.

BACKGROUND: Microbiomes consist of bacteria, viruses, and other microorganisms, and are responsible for many different functions in both organisms and the environment. Past analyses of microbiomes focused on using correlation to determine linear relationships between microbes and diseases. Weak correlations due to nonlinearity between microbe pairs may cause researchers to overlook critical components of the data. With the abundance of available microbiome, we need a method that comprehensively studies microbiomes and how they are related to each other. RESULTS: We collected publicly available datasets from human, environment, and animal samples to determine both symmetric and asymmetric Boolean implication relationships between a pair of microbes. We then found relationships that are potentially invariants, meaning they will hold in any microbe community. In other words, if we determine there is a relationship between two microbes, we expect the relationship to hold in almost all contexts. We discovered that around 330,000 pairs of microbes universally exhibit the same relationship in almost all the datasets we studied, thus making them good candidates for invariants. Our results also confirm known biological properties and seem promising in terms of disease diagnosis. CONCLUSIONS: Since the relationships are likely universal, we expect them to hold in clinical settings, as well as general populations. If these strong invariants are present in disease settings, it may provide insight into prognostic, predictive, or therapeutic properties of clinically relevant diseases. For example, our results indicate that there is a difference in the microbe distributions between patients who have or do not have IBD, eczema and psoriasis. These new analyses may improve disease diagnosis and drug development in terms of accuracy and efficiency.

Helicobacter pylori infection downregulates the DNA glycosylase NEIL2, resulting in increased genome damage and inflammation in gastric epithelial cells.

Infection with the Gram-negative, microaerophilic bacterium Helicobacter pylori induces an inflammatory response and oxidative DNA damage in gastric epithelial cells that can lead to gastric cancer (GC). However, the underlying pathogenic mechanism is largely unclear. Here, we report that the suppression of Nei-like DNA glycosylase 2 (NEIL2), a mammalian DNA glycosylase that specifically removes oxidized bases, is one mechanism through which H. pylori infection may fuel the accumulation of DNA damage leading to GC. Using cultured cell lines, gastric biopsy specimens, primary cells, and human enteroid-derived monolayers from healthy human stomach, we show that H. pylori infection greatly reduces NEIL2 expression. The H. pylori infection-induced downregulation of NEIL2 was specific, as Campylobacter jejuni had no such effect. Using gastric organoids isolated from the murine stomach in coculture experiments with live bacteria mimicking the infected stomach lining, we found that H. pylori infection is associated with the production of various inflammatory cytokines. This response was more pronounced in Neil2 knockout (KO) mouse cells than in WT cells, suggesting that NEIL2 suppresses inflammation under physiological conditions. Notably, the H. pylori-infected Neil2-KO murine stomach exhibited more DNA damage than the WT. Furthermore, H. pylori-infected Neil2-KO mice had greater inflammation and more epithelial cell damage. Computational analysis of gene expression profiles of DNA glycosylases in gastric specimens linked the reduced Neil2 level to GC progression. Our results suggest that NEIL2 downregulation is a plausible mechanism by which H. pylori infection impairs DNA damage repair, amplifies the inflammatory response, and initiates GC.

Fungal effectors, the double edge sword of phytopathogens.

Phyto-pathogenic fungi can cause huge damage to crop production. During millions of years of coexistence, fungi have evolved diverse life-style to obtain nutrients from the host and to colonize upon them. They deploy various proteinaceous as well as non-proteinaceous secreted molecules commonly referred as effectors to sabotage host machinery during the infection process. The effectors are important virulence determinants of pathogenic fungi and play important role in successful pathogenesis, predominantly by avoiding host-surveillance system. However, besides being important for pathogenesis, the fungal effectors end-up being recognized by the resistant cultivars of the host, which mount a strong immune response to ward-off pathogens. Various recent studies involving different pathosystem have revealed the virulence/avirulence functions of fungal effectors and their involvement in governing the outcome of host-pathogen interactions. However, the effectors and their cognate resistance gene in the host remain elusive for several economically important fungal pathogens. In this review, using examples from some of the biotrophic, hemi-biotrophic and necrotrophic pathogens, we elaborate the double-edged functions of fungal effectors. We emphasize that knowledge of effector functions can be helpful in effective management of fungal diseases in crop plants.

Fusobacterium nucleatum promotes colorectal cancer by inducing Wnt/ß-catenin modulator Annexin A1.

Fusobacterium nucleatum, a Gram-negative oral anaerobe, is a significant contributor to colorectal cancer. Using an in vitro cancer progression model, we discover that F. nucleatum stimulates the growth of colorectal cancer cells without affecting the pre-cancerous adenoma cells. Annexin A1, a previously unrecognized modulator of Wnt/ß-catenin signaling, is a key component through which F. nucleatum exerts its stimulatory effect. Annexin A1 is specifically expressed in proliferating colorectal cancer cells and involved in activation of Cyclin D1. Its expression level in colon cancer is a predictor of poor prognosis independent of cancer stage, grade, age, and sex. The FadA adhesin from F. nucleatum up-regulates Annexin A1 expression through E-cadherin. A positive feedback loop between FadA and Annexin A1 is identified in the cancerous cells, absent in the non-cancerous cells. We therefore propose a "two-hit" model in colorectal carcinogenesis, with somatic mutation(s) serving as the first hit, and F. nucleatum as the second hit exacerbating cancer progression after benign cells become cancerous. This model extends the "adenoma-carcinoma" model and identifies microbes such as F. nucleatum as cancer "facilitators".

Enhanced mitochondrial fission suppresses signaling and metastasis in triple-negative breast cancer.

BACKGROUND: Mitochondrial dynamics underlies malignant transformation, cancer progression, and response to treatment. Current research presents conflicting evidence for functions of mitochondrial fission and fusion in tumor progression. Here, we investigated how mitochondrial fission and fusion states regulate underlying processes of cancer progression and metastasis in triple-negative breast cancer (TNBC). METHODS: We enforced mitochondrial fission and fusion states through chemical or genetic approaches and measured migration and invasion of TNBC cells in 2D and 3D in vitro models. We also utilized kinase translocation reporters (KTRs) to identify single cell effects of mitochondrial state on signaling cascades, PI3K/Akt/mTOR and Ras/Raf/MEK/ERK, commonly activated in TNBC. Furthermore, we determined effects of fission and fusion states on metastasis, bone destruction, and signaling in mouse models of breast cancer. RESULTS: Enforcing mitochondrial fission through chemical or genetic approaches inhibited migration, invasion, and metastasis in TNBC. Breast cancer cells with predominantly fissioned mitochondria exhibited reduced activation of Akt and ERK both in vitro and in mouse models of breast cancer. Treatment with leflunomide, a potent activator of mitochondrial fusion proteins, overcame inhibitory effects of fission on migration, signaling, and metastasis. Mining existing datasets for breast cancer revealed that increased expression of genes associated with mitochondrial fission correlated with improved survival in human breast cancer. CONCLUSIONS: In TNBC, mitochondrial fission inhibits cellular processes and signaling pathways associated with cancer progression and metastasis. These data suggest that therapies driving mitochondrial fission may benefit patients with breast cancer.

The PVT1 lncRNA is a novel epigenetic enhancer of MYC, and a promising risk-stratification biomarker in colorectal cancer.

Accumulating evidence suggests that dysregulation of transcriptional enhancers plays a significant role in cancer pathogenesis. Herein, we performed a genome-wide discovery of enhancer elements in colorectal cancer (CRC). We identified PVT1 locus as a previously unrecognized transcriptional regulator in CRC with a significantly high enhancer activity, which ultimately was responsible for regulating the expression of MYC oncogene. High expression of the PVT1 long-non-coding RNA (lncRNA) transcribed from the PVT1 locus was associated with poor survival among patients with stage II and III CRCs (p < 0.05). Aberrant methylation of the PVT1 locus inversely correlated with the reduced expression of the corresponding the PVT1 lncRNA, as well as MYC gene expression. Bioinformatic analyses of CRC-transcriptomes revealed that the PVT1 locus may also broadly impact the expression and function of other key genes within two key CRC-associated signaling pathways - the TGFß/SMAD and Wnt/ß-Catenin pathways. We conclude that the PVT1 is a novel oncogenic enhancer of MYC and its activity is controlled through epigenetic regulation mediated through aberrant methylation in CRC. Our findings also suggest that the PVT1 lncRNA expression is a promising prognostic biomarker and a potential therapeutic target in CRC.

CDX2 as a Prognostic Biomarker in Stage II and Stage III Colon Cancer.

Background The identification of high-risk stage II colon cancers is key to the selection of patients who require adjuvant treatment after surgery. Microarray-based multigene-expression signatures derived from stem cells and progenitor cells hold promise, but they are difficult to use in clinical practice. Methods We used a new bioinformatics approach to search for biomarkers of colon epithelial differentiation across gene-expression arrays and then ranked candidate genes according to the availability of clinical-grade diagnostic assays. With the use of subgroup analysis involving independent and retrospective cohorts of patients with stage II or stage III colon cancer, the top candidate gene was tested for its association with disease-free survival and a benefit from adjuvant chemotherapy. Results The transcription factor CDX2 ranked first in our screening test. A group of 87 of 2115 tumor samples (4.1%) lacked CDX2 expression. In the discovery data set, which included 466 patients, the rate of 5-year disease-free survival was lower among the 32 patients (6.9%) with CDX2-negative colon cancers than among the 434 (93.1%) with CDX2-positive colon cancers (hazard ratio for disease recurrence, 3.44; 95% confidence interval [CI], 1.60 to 7.38; P=0.002). In the validation data set, which included 314 patients, the rate of 5-year disease-free survival was lower among the 38 patients (12.1%) with CDX2 protein-negative colon cancers than among the 276 (87.9%) with CDX2 protein-positive colon cancers (hazard ratio, 2.42; 95% CI, 1.36 to 4.29; P=0.003). In both these groups, these findings were independent of the patient's age, sex, and tumor stage and grade. Among patients with stage II cancer, the difference in 5-year disease-free survival was significant both in the discovery data set (49% among 15 patients with CDX2-negative tumors vs. 87% among 191 patients with CDX2-positive tumors, P=0.003) and in the validation data set (51% among 15 patients with CDX2-negative tumors vs. 80% among 106 patients with CDX2-positive tumors, P=0.004). In a pooled database of all patient cohorts, the rate of 5-year disease-free survival was higher among 23 patients with stage II CDX2-negative tumors who were treated with adjuvant chemotherapy than among 25 who were not treated with adjuvant chemotherapy (91% vs. 56%, P=0.006). Conclusions Lack of CDX2 expression identified a subgroup of patients with high-risk stage II colon cancer who appeared to benefit from adjuvant chemotherapy. (Funded by the National Comprehensive Cancer Network, the National Institutes of Health, and others.).

Identification of tumorigenic cells and therapeutic targets in pancreatic neuroendocrine tumors.

Pancreatic neuroendocrine tumors (PanNETs) are a type of pancreatic cancer with limited therapeutic options. Consequently, most patients with advanced disease die from tumor progression. Current evidence indicates that a subset of cancer cells is responsible for tumor development, metastasis, and recurrence, and targeting these tumor-initiating cells is necessary to eradicate tumors. However, tumor-initiating cells and the biological processes that promote pathogenesis remain largely uncharacterized in PanNETs. Here we profile primary and metastatic tumors from an index patient and demonstrate that MET proto-oncogene activation is important for tumor growth in PanNET xenograft models. We identify a highly tumorigenic cell population within several independent surgically acquired PanNETs characterized by increased cell-surface protein CD90 expression and aldehyde dehydrogenase A1 (ALDHA1) activity, and provide in vitro and in vivo evidence for their stem-like properties. We performed proteomic profiling of 332 antigens in two cell lines and four primary tumors, and showed that CD47, a cell-surface protein that acts as a "don't eat me" signal co-opted by cancers to evade innate immune surveillance, is ubiquitously expressed. Moreover, CD47 coexpresses with MET and is enriched in CD90(hi)cells. Furthermore, blocking CD47 signaling promotes engulfment of tumor cells by macrophages in vitro and inhibits xenograft tumor growth, prevents metastases, and prolongs survival in vivo.

Comparison and storage study of ultra-filtered clarified jamun (Syzygium cumini) juice.

Ultrafiltration is one of the types of clarification process that was undertaken in this study together with examining and comparing the storage period of clarified juice by other methods (enzymatically treated, centrifugation, microfiltration). Centrifugation, ultrafiltration and microfiltration of jamun juice was carried out using a laboratory scale refrigerated centrifuge and filtration system. The juices obtained from various processes were evaluated on the basis of their physiochemical and microbial aspects over a period of 8 weeks. Enzyme treated and centrifuged juices were found to be degraded within 15-30 days while other juices had lesser changes in their properties. However microfiltered juice contained some yeasts and molds which increased with time. Ultrafiltration with 50 kDa pore size and 20 psi pressure was found to be the best method for clarification of jamun juice having a prolong shelf life with optimum qualities.

CD14-expressing cancer cells establish the inflammatory and proliferative tumor microenvironment in bladder cancer.

Nonresolving chronic inflammation at the neoplastic site is consistently associated with promoting tumor progression and poor patient outcomes. However, many aspects behind the mechanisms that establish this tumor-promoting inflammatory microenvironment remain undefined. Using bladder cancer (BC) as a model, we found that CD14-high cancer cells express higher levels of numerous inflammation mediators and form larger tumors compared with CD14-low cells. CD14 antigen is a glycosyl-phosphatidylinositol (GPI)-linked glycoprotein and has been shown to be critically important in the signaling pathways of Toll-like receptor (TLR). CD14 expression in this BC subpopulation of cancer cells is required for increased cytokine production and increased tumor growth. Furthermore, tumors formed by CD14-high cells are more highly vascularized with higher myeloid cell infiltration. Inflammatory factors produced by CD14-high BC cells recruit and polarize monocytes and macrophages to acquire immune-suppressive characteristics. In contrast, CD14-low BC cells have a higher baseline cell division rate than CD14-high cells. Importantly, CD14-high cells produce factors that further increase the proliferation of CD14-low cells. Collectively, we demonstrate that CD14-high BC cells may orchestrate tumor-promoting inflammation and drive tumor cell proliferation to promote tumor growth.