Fraction Of Epithelial Cells Research Articles

The basement membrane regulates the cellular localization and the cytoplasmic interactome of Yes-Associated Protein (YAP) in mammary epithelial cells.

The Hippo pathway, a signaling cascade involved in the regulation of organ size and several other processes, acts as a conduit between extracellular matrix (ECM) cues and cellular responses. We asked whether the basement membrane (BM),a specialized ECM component known to induce quiescence and differentiation in mammary epithelial cells, would regulate the localization, activity, and interactome of YAP, a Hippo pathway effector. To address this question, we used a broad range of experimental approaches, including 2D and 3D cultures of both mouse and human mammary epithelial cells, as well as the developing mouse mammary gland. In contrast to malignant cells, nontumoral cells cultured with a reconstituted BM (rBM)displayed higher concentrations of YAP in the cytoplasm. Incidentally, when in the nucleus of rBM-treated cells, YAP resided preferentially at the nuclear periphery. In agreement with our cell culture experiments, YAP exhibited cytoplasmic predominance in ductal cells of developing mammary epithelia, where a denser BM is found. Conversely, terminal end bud (TEB)cells with a thinner BM displayed higher nucleus-to-cytoplasm ratios of YAP. Bioinformatic analysis revealed that genes regulated by YAP were overrepresented in the transcriptomes of microdissected TEBs. Consistently, mouse epithelial cells exposed to the rBM expressed lower levels of YAP-regulated genes, although the protein level of YAP and Hippo components were slightly altered by the treatment. Mass spectrometry analysis identified a differential set of proteins interacting with YAP in cytoplasmic fractions of mouse epithelial cells in the absence or presence of rBM. In untreated cells, YAP interactants were enriched in processes related to ubiquitin-mediated proteolysis, whereas in cells exposed to rBM YAP interactants were mainly key proteins related to amino acid, amino sugar, and carbohydrate metabolism. Collectively, we unraveled that the BM induces YAP translocation or retention in the cytoplasm of nontumoral epithelial cells and that in the cytoplasm YAP seems to undertake novel functions in metabolic pathways.

P040 Prediction of endoscopic features of disease from single-cell RNA sequencing data in sigmoid colon of patients with ulcerative colitis

Abstract Background Patients suffering from ulcerative colitis (UC) have reduced life qualities and an increased risk of developing cancer. Current treatment options are limited and have a ceiling effect with remission rates of ~35%. A possible reason is the multi-factorial aetiology, which are associated with an overactivation of the immune system and a dysbiosis of the gut microbiome. An important step towards a better understanding is the characterization of epithelial lesions. Their severity is classified by the Mayo score system based on their macroscopic appearance. While previous studies analysed cellular changes within the colon in UC patients, a detailed characterization of different UC-associated lesions are still missing, which is the aim of our study in collaboration with Sanofi Pharmaceuticals. Methods We recruit patients with active disease at our endoscopy unit and collect different biopsies from multiple regions within these patients to compare the reproducibility of single cell RNA sequencing (scRNAseq) results of similar appearing lesions within and between patients. For the sample preparation, we include a cellular enrichment step for immune, epithelial and stromal cells before performing 10x scRNAseq to obtain equal numbers for each cell fraction from every sampled area. Results Our cellular isolation strategy allows successful recovery of fibroblast, epithelial and immune cell fractions from each lesion. All expected epithelial cell types as previously described are detected in our dataset as well. Moreover, our experimental setup to obtain comparable numbers of fibroblasts, epithelial and immune cells enables us now to investigate cell-cell communication differences within different Mayo score lesions. For example, CXCL signal increased with the severity of macroscopic lesions and involves multiple cell types. Conclusion Our preliminary results promise exciting insights into cell-cell communications at an unprecedented depth, which identifies aberrant signalling pathway that may contribute to treatment failure in the clinics.

Abstract A089: Quantitative MRI metrics and single cell transcriptome reveal effects of stroma-directed drug and chemotherapy in a GEM model of pancreatic cancer

Abstract Hypothesis: The dense stroma present in pancreatic ductal adenocarcinoma (PDA) harbors a unique tumor microenvironment (TME) that is immune suppressive and underlies the chemo-resistance of PDA. We have evaluated synthetic vitamin D combined with chemoimmunotherapy in a pilot clinical study (NCT03519308). To elucidate the underlying mechanisms of this treatment, we designed a matched study in a genetically engineered mouse model of PDA (KPC mice) to test the hypothesis that synthetic vitamin D enhances the effect of chemotherapy with or without immune checkpoint inhibition. To detect changes in TME, we have integrated diffusion weighted (DW) and dynamic contrast enhanced (DCE) MRI with immunohistochemistry (IHC) and single cell RNA sequencing (scRNAseq). Methods: KPC mice were randomly assigned to one of four groups: 1) Control (untreated); 2) Nab-paclitaxel + gemcitabine + cisplatin (Chemo); 3) Chemo + synthetic vitamin D (calcipotriol, Cal); 4) Chemo + Cal + PD-L1. The treatment lasted for 14 days followed by euthanasia and tumor collection for IHC and scRNAseq. Respiratory motion-robust DW- and DCE-MRI were performed on treatment days 0, 7 and 14. Results: Compared to consistent tumor growth (measured by MRI) in the Control group, both progression and regression were observed in groups 2) - 4), suggesting that the KPC model captured the varied treatment responses observed in the clinic. Capillary perfusion/permeability of the tumor measured by Ktrans (a DCE-MRI metric) was reduced at day 7 in the Chemo group (P<0.05), whereas Chemo+Cal reversed the reduction of Ktrans. Chemo-induced Ktrans reduction was corroborated with a significant reduction of microvascular density by IHC of CD31 compared to the Control. Addition of PD-L1 did not appear to enhance the tumor growth delay by Chemo+Cal. scRNAseq analysis revealed that while Chemo significantly delayed the tumor growth compared to the Control, it increased the portion of mesenchymal cells in the tumor and upregulated epithelial-to-mesenchymal transition (EMT) genes known to resist chemotherapy. In contrast, Chemo+Cal increased the fraction of epithelial cells and downregulated EMT genes compared to Chemo. Trasncriptome analysis of the tumor cells demonstrated that Chemo treatment increased the expression of Fibronectin 1 (FN1), a gene associated with tumor invasiveness, EMT, and metastasis, while lowering the expression of Trefoil Factor Family1 (Tff1), which is related to tumor-suppression and chemosensitivity. Chemo+Cal treatment reversed the expression of both FN1 and Tff1 gene compared to Chemo treatment. Conclusion: Single cell transcriptome analysis provided valuable insights into treatment-induced molecular subtype changes, which may predict future resistance and invasiveness. Our study revealed important mechanisms of calcipotriol that enhance the efficacy of chemotherapy by improving microvascular perfusion and permeability and by reducing chemo-resistance by enriching epithelial cells in PDA tumors. Citation Format: Hoon Choi, Mamta Gupta, Thomas Karasic, Miguel Joaquim, Emma Furth, Stephen Pickup, Cynthia Clendenin, Hee Kwon Song, Yong Fan, Jeffrey Duda, James Gee, Mark Rosen, Peter O’Dwyer, Rong Zhou. Quantitative MRI metrics and single cell transcriptome reveal effects of stroma-directed drug and chemotherapy in a GEM model of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A089.

Mouse In Vitro Spermatogenesis on 3D Bioprinted Scaffolds.

Testes have a complex architecture that is compartmentalized into seminiferous tubules with a diameter of approximatively 200μm in which the germ cells differentiate, surrounded by a basement membrane and interstitium. 3D bioprinting might be used to recreate the compartmentalized testicular architecture in vitro. Directed by a software program, pneumatic microextrusion printers can deposit 3D layers of hydrogel-encapsulated interstitial cells in a controlled manner by applying pressure. Once macroporous-shaped scaffolds resembling seminiferous tubules have been bioprinted with interstitial cells, the epithelial cell fraction can be seeded in the macropores to resemble the in vivo testicular architecture. Moreover, macropores can serve as a delimitation for all testicular cells to reorganize and improve the supply of nutrients to cells through the 3D constructs.

Vesicular Release and Uptake of Circular LSD1-RNAs from Non-Cancer and Cancer Lung Cells.

Lysine-specific demethylase 1 (LSD1) is highly expressed in many cancer types and strongly associated with cancer progression and metastasis. Circular RNAs (circRNAs) are produced by back-splicing and influence the interactive RNA network by microRNA and protein sponging. In the present study, we aimedto identify circRNAs that derive from the LSD1-encoding KDM1A gene, and to investigate their potential to be released and uptaken by lung cancer versus non-cancer epithelial cells. We identified four circLSD1-RNAs by RT-PCR with divergent primers, followed by sequencing. The expression level of circLSD1-RNAs was then studied by quantitative PCR on cellular and extracellular fractions of lung cancer (PC9) and non-cancer primary small airway epithelial (PSAE) cells. Moreover, we established the transgenic overexpression of circLSD1-RNAs. We show that circLSD1-RNAs are primarily located in the cytoplasm, but are packaged and released from lung cancer and non-cancer cells by extracellular vesicles (EVs) and ribonucleoprotein (RNP) complexes, respectively. Proteomics demonstrated a different protein pattern of EV fractions released from PC9 versus PSAE cells. Importantly, released circLSD1-RNAs were differently taken up by PSAE and PC9 cells. In conclusion, our findings provide primary evidence that circLSD1-RNAs participate in the intercellular communication of lung cancer cells with the tumor environment.

Dependence of Induced Biological Damage on the Energy Distribution and Intensity of Clinical Intra-Operative Radiotherapy Electron Beams.

The survival fraction of epithelial HaCaT cells was analysed to assess the biological damage caused by intraoperative radiotherapy electron beams with varying energy spectra and intensities. These conditions were achieved by irradiating the cells at different depths in water using nominal 6 MeV electron beams while consistently delivering a dose of 5 Gy to the cell layer. Furthermore, a Monte Carlo simulation of the entire irradiation procedure was performed to evaluate the molecular damage in terms of molecular dissociations induced by the radiation. A significant agreement was found between the molecular damage predicted by the simulation and the damage derived from the analysis of the survival fraction. In both cases, a linear relationship was evident, indicating a clear tendency for increased damage as the averaged incident electron energy and intensity decreased for a constant absorbed dose, lowering the dose rate. This trend suggests that the radiation may have a more pronounced impact on surrounding healthy tissues than initially anticipated. However, it is crucial to conduct additional experiments with different target geometries to confirm this tendency and quantify the extent of this effect.

NF-κB drives epithelial-mesenchymal mechanisms of lung fibrosis in a translational lung cell model.

In the progression phase of idiopathic pulmonary fibrosis (IPF), the normal alveolar structure of the lung is lost and replaced by remodeled fibrotic tissue and by bronchiolized cystic airspaces. Although these are characteristic features of IPF, knowledge of specific interactions between these pathological processes is limited. Here, the interaction of lung epithelial and lung mesenchymal cells was investigated in a coculture model of human primary airway epithelial cells (EC) and lung fibroblasts (FB). Single-cell RNA sequencing revealed that the starting EC population was heterogenous and enriched for cells with a basal cell signature. Furthermore, fractions of the initial EC and FB populations adopted distinct pro-fibrotic cell differentiation states upon cocultivation, resembling specific cell populations that were previously identified in lungs of patients with IPF. Transcriptomic analysis revealed active NF-κB signaling early in the cocultured EC and FB, and the identified NF-κB expression signatures were found in "HAS1 High FB" and "PLIN2+ FB" populations from IPF patient lungs. Pharmacological blockade of NF-κB signaling attenuated specific phenotypic changes of EC and prevented FB-mediated interleukin-6, interleukin-8, and CXC chemokine ligand 6 cytokine secretion, as well as collagen α-1(I) chain and α-smooth muscle actin accumulation. Thus, we identified NF-κB as a potential mediator, linking epithelial pathobiology with fibrogenesis.

The crosstalk within the breast tumor microenvironment in type II diabetes: Implications for cancer disparities.

Obesity-driven (type 2) diabetes (T2D), the most common metabolic disorder, both increases the incidence of all molecular subtypes of breast cancer and decreases survival in postmenopausal women. Despite this clear link, T2D and the associated dysfunction of diverse tissues is often not considered during the standard of care practices in oncology and, moreover, is treated as exclusion criteria for many emerging clinical trials. These guidelines have caused the biological mechanisms that associate T2D and breast cancer to be understudied. Recently, it has been illustrated that the breast tumor microenvironment (TME) composition and architecture, specifically the surrounding cellular and extracellular structures, dictate tumor progression and are directly relevant for clinical outcomes. In addition to the epithelial cancer cell fraction, the breast TME is predominantly made up of cancer-associated fibroblasts, adipocytes, and is often infiltrated by immune cells. During T2D, signal transduction among these cell types is aberrant, resulting in a dysfunctional breast TME that communicates with nearby cancer cells to promote oncogenic processes, cancer stem-like cell formation, pro-metastatic behavior and increase the risk of recurrence. As these cells are non-malignant, despite their signaling abnormalities, data concerning their function is never captured in DNA mutational databases, thus we have limited insight into mechanism from publicly available datasets. We suggest that abnormal adipocyte and immune cell exhaustion within the breast TME in patients with obesity and metabolic disease may elicit greater transcriptional plasticity and cellular heterogeneity within the expanding population of malignant epithelial cells, compared to the breast TME of a non-obese, metabolically normal patient. These challenges are particularly relevant to cancer disparities settings where the fraction of patients seen within the breast medical oncology practice also present with co-morbid obesity and metabolic disease. Within this review, we characterize the changes to the breast TME during T2D and raise urgent molecular, cellular and translational questions that warrant further study, considering the growing prevalence of T2D worldwide.

Interleukin 18 Suppresses Regeneration of the Thymus

The thymus is highly sensitive to acute injury, particularly to cytoreductive conditioning given pre-hematopoietic stem cell transplant (HCT). The thymus is capable of a remarkable degree of regeneration, however its reparative capacity and T cell productivity decline with age. This leaves HCT recipients vulnerable to relapse of malignancy and opportunistic infection - the leading causes of post-HCT mortality - during a prolonged period of lymphopenia. Better understanding the endogenous mechanisms by which thymus regeneration is regulated may inform therapeutic interventions to improve T cell reconstitution in these patients. Here, we identify the inflammatory cytokine interleukin 18 (IL-18) as a novel suppressant of thymic function and propose its targeting to improve immune reconstitution. Recently, our group reported that attenuation of homeostatic detection of apoptotic thymocytes after damage regulates tissue regeneration by releasing the brakes on the production of the regenerative factors IL-23 and BMP4 (Kinsella 2021 Cell Rep 37:1). However, our preliminary data also shows that upon acute damage by ionizing radiation, there is a switch towards an immunogenic pyroptotic cell death with an increase in cleaved caspase 1 (pyroptotic caspase) and release of damage-associated molecular patterns. Among these was the release of the caspase 1 dependent inflammatory factors IL-1b and IL-18 within the thymus following damage. Although mice deficient for IL-1b receptor (Il1r-/-) did not show any modulation in their ability to regenerate after total body irradiation (TBI), we found that mice deficient for IL-18 (Il18-/-) exhibited increased thymic cellularity (Fig. 1A). Based on these findings, we hypothesized that immunogenic cell death-induced IL-18 suppresses thymic repair following acute insult and that IL-18 can be targeted to improve T cell reconstitution in settings of lymphopenia. Although IL-18R was expressed by a substantial fraction of medullary thymic epithelial cells (mTECs), and TECs serve as the master regulators of thymic function, mice with a deletion for IL-18R specifically in TECs (using Il18r1fl/flFoxn1Cre+ mice) did not show any altered regenerative capacity compared to control mice, suggesting that IL-18 does not act directly on TECs. Instead, we determined most IL-18Rexpression was restricted to NK1.1+ Innate Lymphoid Cells. Strikingly, in vivo depletion of this population prior to induction of damage significantly improved thymic regeneration (Fig. 1B), consistent with what we observed in IL-18 deficient mice. Importantly, we found that thymic NK cells were activated after damage, with increased expression of Perforin, Gzma, and Gzmb, as well as the activation markers Nkg7, Klrd1, and Klrk1 (NKG2D). Suggestive of a mechanism that could be mediating this, mice with a germline deletion for Perforin showed improved thymus repair following acute damage further supporting an NK cell mediated mechanism of thymic suppression. Finally, mice treated with an antibody against IL-18 demonstrated early signs of improved thymic regeneration and export of new naïve T cells in a mouse model of allogeneic HCT. Taken together, these findings implicate the inflammatory cytokine IL-18, signaling through NK cells, as suppressive to endogenous thymic regeneration. Furthermore, our data suggests that this pathway can be therapeutically targeted to improve T cell reconstitution following cytoreductive therapy and HCT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mapping the Universe of Eph Receptor and Ephrin Ligand Transcripts in Epithelial and Fiber Cells of the Eye Lens.

The eye lens is a transparent, ellipsoid organ in the anterior chamber of the eye that is required for fine focusing of light onto the retina to transmit a clear image. Cataracts, defined as any opacity in the lens, remains the leading cause of blindness in the world. Recent studies in humans and mice indicate that Eph–ephrin bidirectional signaling is important for maintaining lens transparency. Specifically, mutations and polymorphisms in the EphA2 receptor and the ephrin-A5 ligand have been linked to congenital and age-related cataracts. It is unclear what other variants of Ephs and ephrins are expressed in the lens or whether there is preferential expression in epithelial vs. fiber cells. We performed a detailed analysis of Eph receptor and ephrin ligand mRNA transcripts in whole mouse lenses, epithelial cell fractions, and fiber cell fractions using a new RNA isolation method. We compared control samples with EphA2 knockout (KO) and ephrin-A5 KO samples. Our results revealed the presence of transcripts for 12 out of 14 Eph receptors and 8 out of 8 ephrin ligands in various fractions of lens cells. Using specific primer sets, RT-PCR, and sequencing, we verified the variant of each gene that is expressed, and we found two epithelial-cell-specific genes. Surprisingly, we also identified one Eph receptor variant that is expressed in KO lens fibers but is absent from control lens fibers. We also identified one low expression ephrin variant that is only expressed in ephrin-A5 control samples. These results indicate that the lens expresses almost all Ephs and ephrins, and there may be many receptor–ligand pairs that play a role in lens homeostasis.

Abstract 6149: Prognostic tumor microenvironment subtypes in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer in which the tumor microenvironment (TME) is prognostic, with increased tumor-infiltrating lymphocytes predicting a good prognosis. Positive clinical trials of immune checkpoint inhibitors in TNBC further highlight the TME’s importance, but its single-cell composition and relationship to patient survival has not been well described. Therefore, we generated highly-multiplex cyclic immunofluorescence (CyCIF) data from 59 TNBC in tissue microarrays. Our CyCIF dataset served as a discovery cohort; the data included 35 protein markers and clinical outcomes for 18 patients. For a validation cohort, we analyzed 32 markers imaged with multiplex ion beam imaging (MIBI) in 38 TNBC patients (Keren et. al., Cell 2018). In the CyCIF cohort, we used markers’ single-cell mean intensity to generate a Umap manifold, which we clustered with the unsupervised Leiden algorithm. We annotated the resulting 28 cell types as epithelial, immune or stromal. In the MIBI cohort, we similarly clustered and annotated 21 cell types as epithelial, immune or stromal. Next, we clustered the patients based on the fraction of epithelial, immune or stromal cells in the tissue. In the CyCIF cohort, patients fell into three subtypes: epithelial-rich, stroma-rich and immune-rich. The patients in the epithelial-rich group had significantly shorter survival than the other two subtypes (log-rank p-value = 0.0008) and had shorter recurrence-free survival (log-rank p-value = 0.048). In the MIBI cohort, patients fell into the same three subtypes. As in the CyCIF cohort, the epithelial-rich patients had shorter survival than the other subtypes (log-rank p-value = 0.021), although there was no difference in recurrence-free survival (log-rank p-value = 0.47). Finally, we identified protein biomarkers with significantly different expression levels between the subtypes in one or both cohorts. The poor-prognosis, epithelial-rich subtype had higher CD31 expression in both cohorts, and in the CyCIF cohort, Glut1 was elevated, indicating a hypoxic, angiogenic TME. The immune infiltrate was primarily CD68+ macrophages and the stroma was activated, with higher levels of Ki67 in both cohorts. In contrast, the stroma-rich, good prognosis subtype had low immune infiltration, including fewer macrophages, low CD31 expression and a quiescent, non-proliferating stroma. Lastly, the good-prognosis, immune-rich subtype had high T and B cells levels, as well as markers of immune regulation, memory cells, and antigen presentation cells. In conclusion, we generated and analyzed a new multiplex CyCIF dataset with clinical annotation and confirmed our analytical findings in a second multiplex imaging dataset. We delineated three TME subtypes in TNBC with different outcomes, cell types and biomarker expression, potentially leading to better patient stratification and informing new drug targets. Citation Format: Jennifer R. Eng, Elmar Bucher, Zhi Hu, Melinda Sanders, Bapsi Chakravarthy, Summer Gibbs, Koei Chin, Jennifer Pietenpol, Joe W. Gray. Prognostic tumor microenvironment subtypes in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6149.

High Autophagic Vesicle Content Identifies a Novel Slow‐cycling Esophageal Basal Cell Population with Stem‐like Properties

Esophageal squamous epithelium comprises a basal layer of proliferative cells that differentiate in suprabasal layers before luminal desquamation, facilitating tissue renewal. Controversy exists as to whether the esophageal basal cells are functionally homogenous, with each cell having equal potential to proliferate and differentiate, or heterogenous, with subsets of basal cells harboring differential proliferation and differentiation kinetics. To examine the relationship between the stemness/proliferation axis and autophagy in esophageal basal cells, we stained primary basal cells from wild type mice with the autophagic vesicle (AVs)‐identifying dye Cyto‐ID. AV‐high and AV‐low basal esophageal epithelial cell fractions were sorted by fluorescence‐activated cell sorting (Figure 1A) and stem/progenitor function was assessed by organoid formation assay and passaging. Upon initial plating, organoid formation rate (OFR) was decreased in Cyto‐IDhighbasal cells(0.8%±0.11) as compared to their Cyto‐IDlow counterparts (10.0%±0.06; n=3; p<0.001) (Figure 1B). However, passaging revealed that OFR decreased in Cyto‐IDlow cells (0.36% for passage 2) while increasing in Cyto‐IDhigh cells (14.88% for passage 4) (Figure 1B). Additionally, while Cyto‐IDlowbasal cells maintained spherical morphology upon passaging, structures reminiscent of bud‐like intestinal organoids with enhanced self‐renewal capacity emerged in Cyto‐IDhighcells (Figure 1C). These findings suggest that cells with high AV content may serve as a quiescent stem cell population. To examine the gene expression profiles of Cyto‐IDlowand Cyto‐IDhigh cells, we performed RNA‐Seq and identified >7000 differentially expressed genes (p<0.05, log2 fold‐change= 2) (Figure 1D). Ingenuity pathway analysis revealed reduction in Cell Cycle‐associated genes in Cyto‐IDhigh cells and propidium iodide flow cytometry confirmed impaired cell cycle progression with significant depletion of the S and G2/M fractions in Cyto‐IDhighcells (S: 0.5 %; G2/M: 2.2%) as compared to Cyto‐IDlow cells (S: 3.0%; G2/M: 8.3%, n=3, p=<0.05). Finally, we utilized K5‐CreERT2+/‐; Atg7flox/floxmice with tamoxifen‐inducible autophagy impairment in squamous epithelium to evaluate the functional consequences of autophagy depletion upon esophageal epithelial tissue architecture. Atg7 depletion in vivo resulted in hyperkeratinization, consistent with autophagy limiting cell proliferation of esophageal keratinocytes and supporting a functional role for autophagy in regulating stem cell biology and cell fate determination in the esophagus.These studies identify a novel quiescent/slow‐cycling stem‐like population in esophageal epithelium marked by high AV content. Future studies will define the detailed molecular mechanisms through which autophagy supports stemness in esophageal epithelium and determine the functional contribution of autophagy to the pathogenesis of widely prevalent esophageal diseases.

Cyp19a1 is essential for macrophage function and pulmonary tissue repair following airway epithelial injury

CYP19A1 (the aromatase) is a key enzyme in the biosynthesis of estrogens, which have important regulatory functions, including roles as a major mitogenic stimulus for cellular proliferation. Using a new myeloid cell-specific Cyp19a1-null (Cyp19a1-null) mouse model, we tested the role of macrophage CYP19A1 in pulmonary regenerative response to naphthalene-induced bronchiolar epithelial cell injury. Wild-type and Cyp19a1-null mice were treated with naphthalene (200 mg/kg, IP) to induce acute lung injury, or with corn oil as a vehicle control. Mouse lung was examined at 24 hour, 72 hours, or 14 days after the treatment, for extent of acute airway injury, post-injury cellular proliferation, and tissue repair, respectively. At 24 hours, a genotype difference in the extent of naphthalene-induced airway injury was not detected. At 72 hours, the fraction of airway epithelial cells that are positive for the cell proliferation marker Ki67 was higher in naphthalene-treated wild-type mice (40%-70%) than in naphthalene-treated Cyp19a1-null mice (20-30%); whereas the fraction was much lower in corn oil treated control mice (3-5%). At 14 days, the bronchiolar epithelium was recovered to a greater extent in wild-type (~80%) than in the Cyp19a1-null mice (<30%), as indicated by the abundance of Club cells (positive for Club cell secretary protein) in the airway epithelium. Further studies of isolated macrophages indicated that Cyp19a1-null macrophages had reduced phagocytosis (by ~70%) and migration (by ~50%) activities, compared to wild-type controls. Thus, the loss of macrophage CYP19A1negativelyimpacted macrophage function and impeded the post-injury cellular proliferation and tissue repair in the lung airways.

Abstract 3388: Chromatin accessibility patterns in cell-free DNA reveal tumor heterogeneity

Abstract Introduction: Cell-free DNA (cfDNA) is a robust analyte for non-invasively genotyping cancer patients and useful for both detection and disease monitoring. Recent studies have revealed signals in cfDNA fragmentation patterns useful for inferring epigenetic information by analyzing chromatin accessibility patterns (Snyder et al. 2016; Ulz et al. 2017). However, it remains unclear whether it is feasible to robustly predict RNA expression levels for individual genes by using these cfDNA patterns, as might inform clinically relevant challenges in cancer genomics including cancer classification. Methods: We introduce EPIC-Seq (Epigenetic Profile Inference from CfDNA Deep Sequencing), a sequencing assay targeting transcription start sites (TSSs) of selected genes of interest. We adapt this approach for three clinical problems using a panel of ~200 gene TSSs, including genes informative for cell-of-origin classification of diffuse large B-cell lymphoma (DLBCL: ABC vs GCB), for classification of non-small cell lung cancer histologies (NSCLC: LUAD vs LUSC), and for deconvolution of leukocyte composition by CIBERSORTx digital cytometry. We collected plasma samples from 89 cancer patients (45 DLBCL, 44 NSCLC). We profiled cfDNA from all samples by EPIC-Seq, and then assessed its classification performance against established clinical standards. Results: We analyzed cfDNA fragmentation spectra at TSSs genome-wide in relation to transcriptome-wide gene expression levels. In so doing, we identified a novel chromatin accessibility feature at TSSs in cfDNA robustly varying with expression levels. Combining this accessibility feature with other cfDNA features, we developed a predictive model to infer gene-level transcriptional activity using individual TSSs. When comparing the imputed expression levels using this predictive TSS model with ground truth measured by RNA-seq in peripheral blood, we observed surprisingly high transcriptome-wide correlation (R = 0.81; 10-fold CV). We then asked if inferred expression levels from cfDNA could accurately classify lymphoma subtypes and lung cancer tumor histologies. The resulting classifiers achieved AUC of 0.89 and 0.87 for DLBCL COO and NSLSC subtypes, respectively. We also inferred B-cell and lung epithelial fractions in cfDNA by applying CIBERSORTx to deconvolute EPIC-Seq imputed gene expressions in “bulk cfDNA”. The resulting B cell fractions in DLBCL patients were significantly correlated with tumor burden as measured by mean CAPP-Seq VAF levels (R=0.8, P=0.0001). Similarly, the epithelial cell fractions in NSCLC patients were significantly correlated with mean VAFs (R = 0.5, P= 0.01). Conclusions: We show that by analyzing cfDNA fragmentation patterns at transcription start sites, EPIC-Seq accurately predicts tissue and tumor-specific gene expressions in healthy and cancer plasma samples. EPIC-Seq demonstrates the power of cfDNA analyses to survey multiple measures in cancer such as gene expression and tissue-of-origin. Citation Format: Mohammad Shahrokh Esfahani, Mahya Mehrmohamadi, Chloe B. Steen, Emily G. Hamilton, Daniel A. King, Joanne Soo, Charles Macaulay, Michael Jin, David M. Kurtz, Barzin Nabet, Everett Moding, Jacob Chabon, Aaron Newman, Maximilian Diehn, Ash A. Alizadeh. Chromatin accessibility patterns in cell-free DNA reveal tumor heterogeneity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3388.

Recapitulation of prostate tissue cell type-specific transcriptomes by an in vivo primary prostate tissue xenograft model.

Studies of the normal functions and diseases of the prostate request in vivo models that maintain the tissue architecture and the multiple-cell type compartments of human origin in order to recapitulate reliably the interactions of different cell types. Cell type-specific transcriptomes are critical to reveal the roles of each cell type in the functions and diseases of the prostate. A primary prostate tissue xenograft model was developed using fresh human prostate tissue specimens transplanted onto male mice that were castrated surgically and implanted with a device to maintain circulating testosterone levels comparable to adult human males. Endothelial cells and epithelial cells were isolated from 7 fresh human prostate tissue specimens and from primary tissue xenografts established from 9 fresh human prostate tissue specimens, using antibody-conjugated magnetic beads specific to human CD31 and human EpCAM, respectively. Transcriptomes of endothelial, epithelial and stromal cell fractions were obtained using RNA-Seq. Global and function-specific gene expression profiles were compared in inter-cell type and inter-tissue type manners. Gene expression profiles in the individual cell types isolated from xenografts were similar to those of cells isolated from fresh tissue, demonstrating the value of the primary tissue xenograft model for studies of the inter-relationships between prostatic cell types and the role of such inter-relationships in organ development, disease progression, and response to drug treatments.

Role of myeloid cell CYP19A1 in lung airway tissue repair following naphthalene induced epithelial injury in a mouse model

CYP19A1 (the aromatase) is a key enzyme in the biosynthesis of estrogens, which have important regulatory functions, including roles as a major mitogenic stimulus for cellular proliferation. In this study, we are testing the hypothesis that macrophage CYP19A1 can increase estrogen production and/or regulate the function of macrophages at sites of tissue injury, thereby influencing the balance between normal tissue repair and pathogenic outcomes related to abnormal epithelial repair. A myeloid cell (including macrophage) specific Cyp19a1‐null mouse model was generated and characterized. Wild‐type and Cyp19a1‐null mice were treated with naphthalene (200 mg/kg, IP) to induce acute lung injury, or with corn oil as a vehicle control. Mouse lung was examined at 24 hour, 72 hours, or 14 days after the treatment, for extent of acute airway injury, post‐injury cellular proliferation, and tissue repair, respectively. At 24 hours, a genotype difference in the extent of naphthalene‐induced airway injury was not detected. At 72 hours, the fraction of airway epithelial cells that are positive for the cell proliferation marker Ki67 was higher in naphthalene‐treated wild‐type mice (40%–70%) than in naphthalene‐treated Cyp19a1‐null mice (20–30%); whereas the fraction was much lower in corn oil treated control mice (3–5%). At 14 days, the bronchiolar epithelium was completely recovered in wild‐type mice, but only partially (&lt;30%) in the Cyp19a1‐null mice, as indicated by the abundance of Club cells (positive for Clara cell secretary protein) in the airway epithelium. Thus, the loss of macrophage CYP19A1 did not impact the degree of naphthalene‐induced lung injury; but it impeded the post‐injury cellular proliferation and tissue repair in the airways.Support or Funding InformationSupported in part by NIH grant ES020867

Characterization of Pancreatic and Biliary Cancer Stem Cells in Patient-derived Tissue.

Pancreatic ductal adenocarcinoma (PDAC) and extrahepatic cholangio-carcinoma (eCC) represent two cancer entities with devastating prognoses. Despite recent progress in research and treatment, therapy remains challenging. Cancer stem cells (CSCs) have been shown to play an important role in metastasis and chemoresistance. Therefore, CSCs may play a promising role as a potential therapeutic target. A total of 31 patients (23 PDAC, 8 eCC) were included in the study. CSCs were analyzed in a single-cell suspension of tumor samples via fluorescence-activated cell scanning (FACS) with a functional Hoechst 33342 staining as well as a cell surface marker staining of the CSC-panel (CD24, CD44 and EpCAM) and markers to identify fibroblasts, leukocytes and components of the notch signaling pathway. Furthermore, the potential presence of CSCs among primary cancer-associated fibroblasts (CAFs) was assessed using the same FACS-panel. We showed that CSCs are present in patient-derived dissociated tumor tissue. The functional and surface marker profile of CSC-detection did in fact correlate. The amount of CSCs was significantly correlated with tumor characteristics such as a higher UICC stadium and nodal invasion. CSCs were not restricted to the epithelial cell fraction in tumor tissues, which has been verified in independent analysis of primary cell cultures of CAFs. Our study confirms the in vivo presence of CSCs in PDAC and eCC, stating a clinical significance thereof and thus their plausibility as therapeutic targets. In addition, stem-like cells also seem to constitute a part of the CAFs.

Abstract P1-03-02: Histoepigenetic characterization of breast cancer patient derived xenografts (PDX) implicates epithelial-stromal interactions in differential chemotherapy resistance and PDX engraftment

Abstract Background: Epithelial-stromal interactions play a critical role in treatment resistance, and the state of the stroma can be an indicator for a tumor’s resistance profile. We have developed and characterized a large group of breast cancer PDX models (n=69) (Zhang et al.,2013) and have imported PDX from other sources for inclusion into preclinical drug testing trials. These PDX represent the three main subtypes defined clinically by IHC/FISH (i.e. Estrogen Receptor positive, HER2 positive, and “Triple-negative”). Because the epithelium and stroma in PDX are from two different species (human and mouse, respectively) mRNA (and protein) expression levels can be assigned in a species-specific manner and interrogated computationally to divine interactions that may be important for PDX behaviors including treatment response or engraftment behavior. Methods: Fifty “triple negative” PDX were treated with four weekly cycles of either docetaxel (20mg/kg, IP) or carboplatin (50mg/kg, IP) vs. control, and evaluated quantitatively for the change in tumor volume from baseline (~200mm3) after four weeks. Using deep RNA-seq data (~200M reads/sample), we identified interactions between the epithelial and stromal in the PDX models using CASTIN (Komura et al.,2016) and variations in cancer specific pathways. We then employed a machine learning approach to identify ligand-receptor interactions whose differential expression correlated with treatment response to each agent. We also used epigenomic deconvolution (Onuchic et al., 2016) to devise a novel classification method based on the cancer-cell specific epigenomic profiles of TCGA samples, and then used it to classify the PDX with the intent to correlate with treatment response. Results: PDX showed a full range of responses to each agent, from total resistance to complete response. However, several PDX showed differential responses to either docetaxel or carboplatin. We identified variation in the expression patterns of cancer-associated pathways in the epithelial cell fraction of the tumors, including Wnt signaling, as well as pathways involved in hepatic fibrosis. Informative interactions included bidirectional Eph receptor-ephrin signaling which has previously been found to be overexpressed in breast cancer (Vaught et al.,2008). In the epigenomic deconvolution analysis, our classification scheme divided the basal-like samples in TCGA into two groups: a “hot” immune profile-enriched group, and a “cold” or immune profile-deficient group. By applying this classification to the PDX models, unexpectedly, an overwhelming majority of the triple-negative PDX fell into the “cold” basal group. Thus, classification in this manner showed no association with treatment response in the PDX. Conclusions: These results indicate that tumors may respond uniquely to a given chemotherapeutic, and suggest that differential expression of signaling pathways as well as specific ligand-receptor pairs may prove predictive of resistance and could allow for the development of novel therapies targeting these tissue interactions. While our epigenomic deconvolution results showed no association with treatment response, they unexpectedly suggested that success of PDX engraftment may depend on the stromal immune system composition of the primary tumor such that “hot” primary tumors have lower engraftment efficiency in the “cold” stromal environment of immunocompromised mice. Citation Format: Varduhi Petrosyan, Chen Huang, Ramakrishnan R Srinivasan, Lacey E Dobrolecki, Christina Sallas, Alaina N Lewis, Tao Wang, Bing Zhang, Aleksandar Milosavljevic, Michael T Lewis. Histoepigenetic characterization of breast cancer patient derived xenografts (PDX) implicates epithelial-stromal interactions in differential chemotherapy resistance and PDX engraftment [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-03-02.

Analysis of Mutations in K-ras and p53 Genes in Sputum and Plasma Samples.

Sputum and plasma can provide noninvasive materials to investigate biomarkers for cancer detection and diagnosis. Mutations in the K-ras oncogene and p53 suppressor gene have been frequently found in sputum and plasma samples collected not only from lung cancer patients but also in those of patients prior to presenting clinical symptoms of lung cancer, suggesting that they may also provide useful biomarkers from early lung cancer diagnosis. However, the detection of these mutations has been complicated by the fact that they often occur in only a small fraction of epithelial cells among sputum cells, and of cell-free DNA present in plasma. This chapter describes methods to isolate low fraction epithelial cells from sputum and cell-free DNA from plasma samples obtained from lung cancer patients and to identify low fraction K-ras and p53 mutations in these samples.

Differentiation and Proliferation of Intestinal Stem Cells and its Underlying Regulated Mechanisms during Weaning.

Weaning is a stressful event associated with gastrointestinal disorders and increased disease susceptibility. Many studies have reported the changes that happened in the gut of various mammals such as pigs and rats after weaning. These findings suggest that the development of intestinal tract mainly is affected at the time of weaning through interfering in the differentiation and proliferation of intestinal stem cells. Weaning stress stimulates the rapid differentiation and proliferation of intestinal stem cells in order to adjust to changes caused by weaning, which are mainly manifested as deeper crypt depth and decreased intestine villus height. However, the accelerated cellular process may lead to an increase in the proportion of immature intestinal epithelial cells and goblet cells, which affect intestinal permeability and reduce the gut-barrier function against toxins and pathogens. This review briefly describes the effects coforticotrophin-releasing factor (CRF), epidermal growth factor (EGF) and polyamines on the differentiation and proliferation of intestinal stem cells after weaning and discusses its possible underlying regulatory mechanisms. Firstly, weaning stress activates CRF to binds its receptors, which induces proinflammatory responses and promote rapid differentiation and proliferation of intestinal stem cells to a larger fraction of immature intestinal epithelial cells and goblet cells. Secondly, the lack of EGF after weaning inhibits the expression of goblet cell maturation factors and makes it difficult for goblet cells and intestinal epithelial cells to mature. Finally, diet and endogenous synthesis lead to excessive polyamines in the intestine, which promote the proliferation of intestinal stem cells by regulating the expression of human antigen R (HuR) and other related genes at the time of weaning.