Epithelial Cell Populations Research Articles

Abstract PR06: Cooperative HPV and YAP-mediated oncogenic reprogramming of oral progenitors into cancer stem cells at single cell resolution

Abstract Human papillomavirus-positive head and neck squamous cell carcinoma (HPV+HNSC) constitutes the most common HPV-driven malignancy in the United States, and yet the molecular and cellular mechanisms underlying carcinogenesis in HPV+HNSC remain poorly understood. The decades long latency period and incomplete penetrance linking oral HPV infection to HPV+HNSC suggest that oncogenic events beyond E6-E7 expression are required for carcinogenesis. YAP is a transcriptional co-activator that coordinates cell stemness, growth, proliferation, and survival. We previously identified alterations resulting in YAP activation as a potential oncogenic event in HNSC. We thus hypothesized YAP activation in the context of E6-E7 expression may represent a key tumor initiating event leading to HPV+HNSC. We developed a genetically engineered murine system to investigate the effects of local induction of YAP activation and E6-E7-expression in Keratin14+ (K14+) oral epithelial progenitor cells (OEPCs). In this system, intralingual administration of tamoxifen and dietary administration of doxycycline enables spatiotemporally controlled expression of E6-E7 and/or YAP. Employing this model, we identify combined E6-E7 and YAP activation (E+Y) as sufficient to induce highly penetrant carcinoma formation as early as 10 days after transgene induction. Immunofluorescent analyses showed E+Y resulted in the ectopic expansion of a mitotically active (Ki67+), stem-like (TP63+, SOX2+) epithelial cell population with invasive potential. Bulk RNAseq of microdissected tongue epithelia showed that E+Y uniquely induced transcriptional programs promoting cell cycle progression, epithelial cell plasticity, and acute inflammatory response, which were not observed upon sole activation of either the E6-E7 or YAP transgene. scRNAseq of transgene-induced tongue epithelial cell suspensions identified 8 distinct clusters, including previously defined physiologic oral epithelial cell states and novel transgene-associated clusters. E+Y resulted in the emergence of a unique epithelial cell cluster with transcriptional characteristics consistent with a cancer stem cell population, with enrichment in YAP, mTORC1, E2F, and MYC-driven programs, and pan-cancer cell states including cell cycle, hypoxia, squamous differentiation, and partial EMT. Implantation of E+Y cells resulted in tumor formation in 100% of NSG mice, indicating that E+Y drives the emergence of a cancer stem cell population. We next defined transcriptionally co-expressed modules driving E+Y tumor initiating capacity. Among TCGA-HNSC subjects, 8 of 12 modules were enriched in tumor compared to matched normal tissue. Interestingly, 3 modules (G1/S, cell migration, and focal adhesion) were associated with overall and disease-free survival. Our findings show YAP activation in the context of E6-E7 expression is sufficient for rapid carcinogenesis. We illuminate cellular processes driving malignant conversion at single cell resolution, and show enrichment of these processes in human HNSC exhibiting poor prognosis. Citation Format: Farhoud Faraji, Sydney I. Ramirez, Lauren M. Clubb, Kuniaki Sato, Paola Y. Anguiano Quiroz, William M. G. Galloway, Thomas S. Hoang, Kate Medetgul-Ernar, Pauline Marangoni, Alfredo A. Molinolo, Joseph A. Califano, Quinton Smith, Ophir D. Klein, Pablo Tamayo, J. Silvio Gutkind. Cooperative HPV and YAP-mediated oncogenic reprogramming of oral progenitors into cancer stem cells at single cell resolution [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PR06.

The emerging roles of deep crypt secretory cells in colonic physiology.

Deep crypt secretory (DCS) cells are a population of epithelial cells located at the colonic crypt base that share some similarities to Paneth and goblet cells. They were initially defined as c-Kit expressing cells, though subsequent work showed that they are more specifically marked by Reg4 in the murine colon. The best-understood function of DCS cells at present is supporting the stem cell niche by generating Notch and EGF ligands. However, as these cells also express immunoregulatory (e.g., Ccl6) and host defense (e.g., Retnlb) genes, it is likely they have additional functions in maintaining colonic health outside of maintenance of the stem niche. Recent advances in single-cell transcriptomic profiling hint at additional epithelial and immune roles that may exist for these cells and have aided in elucidating their developmental lineage. This review highlights the emerging evidence supporting a crucial role for DCS cells in intestinal physiology, the current understanding of how these cells are regulated, and their potential role(s) in colonic disease.

Single-cell transcriptomic analysis uncovers the origin and intratumoral heterogeneity of parotid pleomorphic adenoma

Pleomorphic adenoma (PA) is the most common benign tumour in the salivary gland and has high morphological complexity. However, the origin and intratumoral heterogeneity of PA are largely unknown. Here, we constructed a comprehensive atlas of PA at single-cell resolution and showed that PA exhibited five tumour subpopulations, three recapitulating the epithelial states of the normal parotid gland, and two PA-specific epithelial cell (PASE) populations unique to tumours. Then, six subgroups of PASE cells were identified, which varied in epithelium, bone, immune, metabolism, stemness and cell cycle signatures. Moreover, we revealed that CD36+ myoepithelial cells were the tumour-initiating cells (TICs) in PA, and were dominated by the PI3K-AKT pathway. Targeting the PI3K-AKT pathway significantly inhibited CD36+ myoepithelial cell-derived tumour spheres and the growth of PA organoids. Our results provide new insights into the diversity and origin of PA, offering an important clinical implication for targeting the PI3K-AKT signalling pathway in PA treatment.

Cocaine and amphetamine regulated transcript (CART): a newly characterized neuropeptide in human prostate

ABSTRACT Cocaine and amphetamine regulated transcript (CART) is a somatostatin-like polypeptide. CART has been localized in the CNS, hypothalamo-pituitary-adrenocortical (HPA) axis, pancreatic islets and enteric nervous system. We investigated the cellular localization of CART in normal human prostate, benign prostatic hyperplasia, prostatic intraepithelial neoplasia and acinar adenocarcinoma. CART was assessed using immunohistochemistry (IHC) and in situ hybridization (ISH), and its gene expression was identified by RTqPCR. We found cellular expression of CART in both normal prostatic luminal secretory epithelial cells neuroendocrine cells (NEC) of both ducts and acini. The cellular appearance indicated a cycle of neuropeptide synthesis and secretion as validated by ISH/IHC concordance. RTqPCR analysis also validated the immunohistochemical data and gene expression, which both indicated low to moderate expression in prostatic tissues. CART expression also was increased in both neuroendocrine and glandular epithelial cell populations from samples of benign prostatic hyperplasia as validated by IHC, ISH and RTqPCR. CART expression was markedly diminished and, in some cases, entirely absent in tissues of prostatic intraepithelial neoplasia and adenocarcinoma. Owing to loss of CART expression in adenocarcinoma and its increase in benign prostatic hyperplasia, CART may prove to be an important prostate marker.

Method for Collecting Single Epithelial Cells from the Mouse Larynx.

The larynx is lined by specialized epithelial cell populations. Studying molecular changes occurring in individual epithelial cell types requires a reliable method for removing these cells from the larynx. Our objective was to develop a method to harvest individual epithelial cells from the mouse larynx while minimizing contamination from non-laryngeal sites and non-epithelial laryngeal cells. Mice were euthanized, and the larynx was carefully exposed and separated from non-laryngeal sites. A small dental brush was inserted into the laryngeal inlet and rotated to obtain epithelial cells. Cells were transferred to collection media, counted, and cytospin preparations stained for laryngeal epithelial (i.e., Pan-Keratin, EpCAM, NGFR, p63, K5, β-tubulin, MUC5AC) and non-epithelial (i.e., vimentin) cell markers. Histopathology was completed on brushed laryngeal tissue sections to evaluate the depth of cell collection. Preliminary Single-cell RNA sequencing (scRNA-seq) was performed to confirm this method can capture diverse laryngeal cell types. We collected 6000-8000 cells from a single larynx and 35000-40000 cells from combining brushings from three tissues. Histopathology demonstrated brushing removed the epithelial layer of the larynx and some underlying tissue. Immunofluorescence staining demonstrated the phenotype of harvested cells was primarily epithelial. Preliminary scRNA-seq was successfully conducted and displayed nine unique cell clusters. We developed a reliable method of harvesting individual epithelial cells from the mouse larynx. This method will be useful for collection of laryngeal cells for a variety of downstream cellular and molecular assays, including scRNA-seq, protein analyses, and cell-culture-based experiments, following laryngeal injury. NA Laryngoscope, 134:786-794, 2024.

Small extracellular vesicles delivering lncRNA WAC-AS1 aggravate renal allograft ischemia‒reperfusion injury by inducing ferroptosis propagation.

Ferroptosis is a predominant contributor to renal ischemia reperfusion injury (IRI) after kidney transplant, evoking delayed graft function and poorer long-term outcomes. The wide propagation of ferroptosis among cell populations in a wave-like manner, developing the "wave of ferroptosis" causes a larger area of tubular necrosis and accordingly aggravates renal allograft IRI. In this study, we decipher a whole new metabolic mechanism underlying ferroptosis and propose a novel spreading pathway of the "wave of ferroptosis" in the renal tissue microenvironment, in which renal IRI cell-secreted small extracellular vesicles (IRI-sEVs) delivering lncRNA WAC-AS1 reprogram glucose metabolism in adjacent renal tubular epithelial cell populations by inducing GFPT1 expression and increasing hexosamine biosynthesis pathway (HBP) flux, and consequently enhances O-GlcNAcylation. Additionally, BACH2 O-GlcNAcylation at threonine 389 in renal tubular epithelial cells prominently inhibits its degradation by ubiquitination and promotes importin α5-mediated nuclear translocation. We present the first evidence that intranuclear BACH2 suppresses SLC7A11 and GPX4 transcription by binding to their proximal promoters and decreases cellular anti-peroxidation capability, accordingly facilitating ferroptosis. Inhibition of sEV biogenesis and secretion by GW4869 and knockout of lncRNA WAC-AS1 in IRI-sEVs both unequivocally diminished the "wave of ferroptosis" propagation and protected against renal allograft IRI. The functional and mechanistic regulation of IRI-sEVs was further corroborated in an allograft kidney transplant model and an in situ renal IRI model. In summary, these findings suggest that inhibiting sEV-mediated lncRNA WAC-AS1 secretion and targeting HBP metabolism-induced BACH2 O-GlcNAcylation in renal tubular epithelial cells may serve as new strategies for protecting against graft IRI after kidney transplant.

Vulval apocrine adenocarcinoma with tumour-associated eosinophilia in a cat

A 10-year-old, spayed female, Domestic Shorthaired cat was referred for surgical removal of a mass on the left vulval fold. An impression smear revealed mixed cell inflammation, with eosinophils predominating focally, and a concurrent bacterial infection, suggesting a primarily inflammatory lesion. However, cytology of a fine-needle aspirate of the mass revealed a neoplastic epithelial cell population, confirmed on histopathology as an apocrine vulval adenocarcinoma with lymphatic invasion and marked tumour-associated tissue eosinophilia. One month after surgical excision of the mass, the cat developed inguinal metastatic lymphadenopathy and chemotherapy was initiated. The patient ultimately developed marked peripheral lymphadenomegaly and was euthanized due to concerns for overall quality of life and comfort. This case highlights that neoplasia should be a consistent differential diagnosis for eosinophilic infiltrates/inflammation. The distinct appearance of the two cytological samples in this case stresses the need for sampling of different sites of a lesion and the importance of not relying on superficial impression smears for clinical management and prognosis.

Matrix obstructions cause multiscale disruption in collective epithelial migration by suppressing leader cell function.

During disease and development, physical changes in extracellular matrix cause jamming, unjamming, and scattering in epithelial migration. However, whether disruptions in matrix topology alter collective cell migration speed and cell-cell coordination remains unclear. We microfabricated substrates with stumps of defined geometry, density, and orientation, which create obstructions for migrating epithelial cells. Here, we show that cells lose their speed and directionality when moving through densely spaced obstructions. Although leader cells are stiffer than follower cells on flat substrates, dense obstructions cause overall cell softening. Through a lattice-based model, we identify cellular protrusions, cell-cell adhesions, and leader-follower communication as key mechanisms for obstruction-sensitive collective cell migration. Our modeling predictions and experimental validations show that cells' obstruction sensitivity requires an optimal balance of cell-cell adhesions and protrusions. Both MDCK (more cohesive) and α-catenin-depleted MCF10A cells were less obstruction sensitive than wild-type MCF10A cells. Together, microscale softening, mesoscale disorder, and macroscale multicellular communication enable epithelial cell populations to sense topological obstructions encountered in challenging environments. Thus, obstruction-sensitivity could define "mechanotype" of cells that collectively migrate yet maintain intercellular communication.

Increased CD8+ tissue resident memory T cells, regulatory T cells and activated natural killer cells in systemic sclerosis lungs.

Multiple observations indicate a role for lymphocytes in driving autoimmunity in SSc. While T and NK cells have been studied in SSc whole blood and bronchoalveolar lavage fluid, their role remains unclear, partly because no studies have analysed these cell types in SSc-interstitial lung disease (ILD) lung tissue. This research aimed to identify and analyse the lymphoid subpopulations in SSc-ILD lung explants. Lymphoid populations from 13 SSc-ILD and 6 healthy control (HC) lung explants were analysed using Seurat following single-cell RNA sequencing. Lymphoid clusters were identified by their differential gene expression. Absolute cell numbers and cell proportions in each cluster were compared between cohorts. Additional analyses were performed using pathway analysis, pseudotime and cell ligand-receptor interactions. Activated CD16+ NK cells, CD8+ tissue resident memory T cells and Treg cells were proportionately higher in SSc-ILD compared with HC lungs. Activated CD16+ NK cells in SSc-ILD showed upregulated granzyme B, IFN-γ and CD226. Amphiregulin, highly upregulated by NK cells, was predicted to interact with epidermal growth factor receptor on several bronchial epithelial cell populations. Shifts in CD8+ T cell populations indicated a transition from resting to effector to tissue resident phenotypes in SSc-ILD. SSc-ILD lungs show activated lymphoid populations. Activated cytotoxic NK cells suggest they may kill alveolar epithelial cells, while their expression of amphiregulin suggests they may also induce bronchial epithelial cell hyperplasia. CD8+ T cells in SSc-ILD appear to transition from resting to the tissue resident memory phenotype.

Electron, Photon, and Neutron Dose Conversion Coefficients of Lens and Non-Lens Tissues Using a Multi-Tissue Eye Model to Assess Risk of Cataracts and Retinitis.

Previous publications describe the estimation of the dose from ionizing radiation to the whole lens or parts of it but have not considered other eye tissues that are implicated in cataract development; this is especially critical for low-dose, low-ionizing-density exposures. A recent review of the biological mechanisms of radiation-induced cataracts showed that lenticular oxidative stress can be increased by inflammation and vascular damage to non-lens tissues in the eye. Also, the radiation oxygen effect indicates different radiosensitivities for the vascular retina and the severely hypoxic lens. Therefore, this study uses the Monte Carlo N-Particle simulations to quantify dose conversion coefficients for several eye tissues for incident antero-posterior exposure to electrons, photons, and neutrons (and the tertiary electron component of neutron exposure). A stylized, multi-tissue eye model was developed by modifying a model by Behrens etal. (2009) to include the retina, uvea, sclera, and lens epithelial cell populations. Electron exposures were simulated as a single eye, whereas photon and neutron exposures were simulated employing two eyes embedded in the ADAM-EVA phantom. For electrons and photons, dose conversion coefficients are highest for either anterior tissues for low-energy incident particles or posterior tissues for high-energy incident particles. Neutron dose conversion coefficients generally increase with increasing incident energy for all tissues. The ratio of the absorbed dose delivered to each tissue to the absorbed dose delivered to the whole lens demonstrated the considerable deviation of non-lens tissue doses from lens doses, depending on particle type and its energy. These simulations demonstrate that there are large variations in the dose to various ocular tissues depending on the incident radiation dose coefficients; this large variation will potentially impact cataract development.

Identification of BST2 as a conjunctival epithelial stem/progenitor cell marker

The conjunctival epithelium consists of conjunctival epithelial cells and goblet cells derived from conjunctival epithelial stem/progenitor cells. However, the source of these cells is not well known because no specific markers for conjunctival epithelial stem/progenitor cells have been discovered. Therefore, to identify conjunctival epithelial stem/progenitor cell markers, we performed single-cell RNA sequencing of a conjunctival epithelial cell population derived from human-induced pluripotent stem cells (hiPSCs). The following conjunctival epithelial markers were identified: BST2, SLC2A3, AGR2, TMEM54, OLR1, and TRIM29. Notably, BST2 was strongly positive in the basal conjunctival epithelium, which is thought to be rich in stem/progenitor cells. Moreover, BST2 was able to sort conjunctival epithelial stem/progenitor cells from hiPSC-derived ocular surface epithelial cell populations. BST2-positive cells were highly proliferative and capable of successfully generating conjunctival epithelial sheets containing goblet cells. In conclusion, BST2 has been identified as a specific marker of conjunctival epithelial stem/progenitor cells.

CDH12 and the development of the neuronal subtype in MIBC.

e16579 Background: We discovered a novel cell epithelial cell population we termed “C3” in muscle-invasive bladder cancer (MIBC) that is negatively prognostic for surgery and chemotherapy. This cell population shares similarities with the neuronal subtype MIBC (NEBC) which has limited treatment options and is driven by unclear molecular mechanisms. The objective of the study was to determine if NEBC is driven by some of the genes that are overexpressed in C3 cells. A top overexpressed and C3 cell specific gene that is also enriched in normal neuronal tissue is Cadherin 12 (N-Cadherin 2, CDH12). Hence, we initiated a study of this gene. Methods: We overexpressed CDH12 in two murine MIBC cell lines (NA13 and MB49) and then inoculated them into mice. Tumor growth kinetics were studied, and single-nucleus RNA sequencing (snRNA-seq) and bulk RNA sequencing were then used to evaluate the tumors. Tissue samples from patients with MIBC were also analyzed using snRNA-seq and Co-detection by Indexing (CODEX) multiplexed immunofluorescence, and our bioinformatics analysis combined transcriptomic profiles from > 2000 MIBC samples from published datasets, to characterize the transcriptional programs induced by enforcing CDH12 expression. Results:Enforced expression of CDH12 led to enhanced tumor growth and the appearance of the neuroendocrine tumor phenotype. NEBC from human samples showed the highest expression of CDH12, and snRNA-seq results showed that NEBC also has the highest proportion of CDH12 epithelial cells and demonstrated co-expression of CDH12 and neuronal markers (NCAM1, CHGA, and SYP). RNA velocity analysis in murine and human tumors implicated CDH12 as a driver for the development of the neuronal subtype. Conclusions: To our knowledge, this study provides the first identification of a genetic driver leading to NEBC and implicates a novel gene, CDH12 in this process.

The cellular and immunological dynamics of early and transitional human milk

Human milk is essential for infant nutrition and immunity, providing protection against infections and other immune-mediated diseases during the lactation period and beyond in later childhood. Milk contains a broad range of bioactive factors such as nutrients, hormones, enzymes, immunoglobulins, growth factors, cytokines, and antimicrobial factors, as well as heterogeneous populations of maternal cells. The soluble and cellular components of milk are dynamic over time to meet the needs of the growing infant. In this study, we utilize systems-approaches to define and characterize 62 analytes of the soluble component, including immunoglobulin isotypes, as well as the cellular component of human milk during the first two weeks postpartum from 36 mothers. We identify soluble immune and growth factors that are dynamic over time and could be utilized to classify milk into different phenotypic groups. We identify 24 distinct populations of both epithelial and immune cells by single-cell transcriptome analysis of 128,016 human milk cells. We found that macrophage populations have shifting inflammatory profiles during the first two weeks of lactation. This analysis provides key insights into the soluble and cellular components of human milk and serves as a substantial resource for future studies of human milk.

Epigenetic plasticity cooperates with cell-cell interactions to direct pancreatic tumorigenesis.

The response to tumor-initiating inflammatory and genetic insults can vary among morphologically indistinguishable cells, suggesting as yet uncharacterized roles for epigenetic plasticity during early neoplasia. To investigate the origins and impact of such plasticity, we performed single-cell analyses on normal, inflamed, premalignant, and malignant tissues in autochthonous models of pancreatic cancer. We reproducibly identified heterogeneous cell states that are primed for diverse, late-emerging neoplastic fates and linked these to chromatin remodeling at cell-cell communication loci. Using an inference approach, we revealed signaling gene modules and tissue-level cross-talk, including a neoplasia-driving feedback loop between discrete epithelial and immune cell populations that was functionally validated in mice. Our results uncover a neoplasia-specific tissue-remodeling program that may be exploited for pancreatic cancer interception.

Abstract 1726: Loss of NF1 drives hormone dependent mammary carcinogenesis in a rat model with intact immune system

Abstract Loss of NF1 plays a major role as an oncogenic driver in up to 33% of all breast cancers (BC). Loss of NF1 is also a prognostic indicator for increased cancer risk at an earlier age, poorer outcomes, and therapeutic resistance. NF1 has recently been shown to bind the estrogen receptor alpha (ER) and act as a transcriptional corepressor. This helps explain findings in ER+ BC. Changes to NF1 that abrogate ER signaling thereby lead to Ras driven tumor resistance to endocrine therapy as cells are able to grow in low levels of E2 (and tamoxifen). Hence, NF1-mutant tumors represent a distinct molecular class in need of new therapeutics targeting both Ras and ER pathways. We have generated preclinical mammalian models of NF1 loss and BC that can be used to evaluate the role of NF1/ER transcriptional signaling in BC, the role of immune cells in BC, and therapeutics. Our models include a pathogenic patient missense allele c.3827G>A, p.R1276Q (knockin or KI) as well as a 14 base pair deletion c.3661_3674del, p.P1220fs*1223 (knockout or KO) model. Heterozygous (het) Nf1 female rats develop mammary gland adenocarcinoma spontaneously, but het KO rats develop multiple tumors with earlier onset. Tumors are generally Grade 2 and do not differ by genotype. By 16 weeks, 70% of KO females have developed at least one tumor whereas only 20% of KI rats and 2% of WT rats have developed tumors. This impacts survival as 76% of KI and 58% of KO females survive to one year. By 2 years, both alleles have 54% survival. The divergence in phenotype between KI and KO alleles may be due to residual function of R1276Q missense NF1 protein. A more in-depth analysis indicates that mammary tumor formation likely begins relatively early, as we find evidence of aberrant morphology and hyperplasia prior to the formation of palpable tumors. Interestingly, we find histological evidence of lung metastases and expression of breast markers GCDFP15, MGA, and CEA in the lung. Again, we see allele-specific effects in that KO rats develop lung tumors earlier than KI rats. Using single nuclei RNASeq to characterize the transcriptional profile of the mammary tumors, we find allele-specific effects beyond repression of Ras activity that drive aggressive tumor development. We identified different tumor cell populations (2 epithelial cell populations, Myeloid cells, B cells, T cells, Basal mammary cells, and WT specific cells) and identified different pathways altered due to the loss of Nf1 including Ephrin B signaling, Cyclin and Cell Cycle signaling, and Glycolysis signaling. Our overall goal is to characterize the phenotype of these rat models in terms of histopathology, Ras signaling, hormone signaling, immune components, and targeted drug response; which can then be compared with what is known regarding patients with somatic or germline inactivation of NF1 and breast cancer. Ultimately, this will provide better prognostic predictions for patients and better therapeutic options. Citation Format: Deeann Wallis, Christian Fay, Kelley Bradley, Erik Westin, William Bradley, Hui Liu, David Crossman, Jeremy B. Foote, Robert Kesterson. Loss of NF1 drives hormone dependent mammary carcinogenesis in a rat model with intact immune system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1726.

Abstract 4235: Obesity drives inflammation and metabolic reprogramming in an orthotopic mouse model of early colon cancer

Abstract Obesity is strongly associated with colon cancer incidence and, in animal models, promotes colon cancer growth and progression. Mounting evidence indicates that circulating growth factors and chronic inflammation are procancer factors likely mediating this relationship. While obesity blunts antitumor immunity in many cancer types, how obesity-driven inflammation and reduced antitumor immunity may cooperate to promote colon cancer progression is not yet clear. Here we used a murine syngeneic orthotopic transplantation model of colon cancer to profile changes driven by diet-induced obesity (DIO) in the immune and cancer cell compartments of the tumor using digital spatial profiling and RNA sequencing. 7-9-week-old male and female C57BL/6 mice remained on either a 10 kcal% fat diet (control, n=34) or a 60 kcal% fat diet (DIO, n=27) for at least 19 weeks. 700 Apc-null;KrasG12D/+;Trp53-null;Smad4-null;tdTomato (i.e., AKPS) organoids were transplanted into the colonic wall of control and DIO mice via colonoscopy-guided injection. Tumors were excised after four weeks of growth, an early time point to assess changes between diet groups independent of tumor size differences or weight loss associated with tumor growth. RNA sequencing was performed on RNA extracted from tumoral CD45+ and EpCAM+ cell fractions isolated using Milltenyi MicroBeads. DIO mice weighed more than controls (males: controls 37.0 g ± 5.1 vs. DIO 47.4 g ± 4.3; females: controls 26.4 g ± 3.7 vs. DIO 45.9 g ± 9.7) and had more mesenteric fat surrounding the colon (males: controls 388 mg ± 217 vs. DIO 577 mg ± 185; females: controls 201 mg ± 143 vs 1176 mg ± 589). Leptin, resistin, and plasminogen activator inhibitor-1 were significantly elevated in the serum of DIO mice relative to control mice. Tumor weight did not differ between groups. Gene set enrichment analysis demonstrated that tumor cells from DIO mice were significantly enriched for pathways related to inflammation (INFLAMMATORY_RESPONSE, IL6_JAK_STAT3_SIGNALING), glucose and lipid metabolism (GLYCOLYSIS, FATTY_ACID_METABOLISM), and proliferation (E2F_TARGETS, G2M_CHECKPOINT). Within the CD45+ transcriptomic profiles, Tumor Immune Estimation Resource (TIMER2.0) indicated an increase in M0- and M1-like macrophages and B cells in tumors from DIO compared with control mice. Analysis of whole tumor sections from DIO mice showed significantly reduced CD3+ cells by immunofluorescence and more Ki67+ CD45+ immune cells by digital spatial profiling relative to tumors from control mice. Taken together, obesity drives an inflammatory profile in both the immune and epithelial cell populations of AKPS murine tumors, which is associated with increased markers of proliferation. Future studies will address whether inflammatory signals from the mesenteric fat adjacent to the colon tumors may contribute to obesity-driven tumor inflammation, and thereby to colon cancer progression. Citation Format: Elaine M. Glenny, Babak Mirminachi, Tori L. McFarlane, Jatin Roper, Stephen D. Hursting. Obesity drives inflammation and metabolic reprogramming in an orthotopic mouse model of early colon cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4235.

Abstract 5633: Analysis of the immune microenvironment and tumor-infiltrating immune cells across different solid tumors by combined spatial transcriptomics and proteomics

Abstract With the advent of immunotherapies such as immune checkpoint inhibitors and CAR T cells, the interest in the tumor microenvironment and tumor-infiltrating lymphocytes (TILs) cells has increased in recent years as these play a critical role in predicting response to therapy and clinical outcomes. We have reported on the identification of potential targets for CAR T cell therapies by analyzing a multitude of tumor samples with hundreds of antibodies by flow cytometry and a new spatial proteomics platform, the MACSima Imaging Platform (Schäfer et al. Nat. Commun., 2021; Kinkhabwala et.al. Sci. Rep., 2022). Here we report on the analysis of tumor microenvironment and tumor infiltrating immune cells using a novel combined spatial transcriptomics and proteomics approach. For protein expression analysis we generated a panel of 60 fluorochrome conjugated antibodies (REAscreen Immuno-oncology, human, FFPE, version 01) selected to identify immune cells, cancer associated fibroblasts, matrix, blood vessels, lymphatics and malignant epithelial cell populations. RNA analysis was based on a novel in situ hybridization and amplification method followed by a cyclic spatial decoding of transcripts (RNAsky). To standardize and further ease the process of protein and RNA detection we generated the antigen and RNA detection probes in a ready-to-use format, i.e., dried and sealed in 96 well plates ready to be inserted into the MACSima. FFPE specimens of different tumor entities reviewed by a pathologist were first hybridized to probes, which were amplified, and spatially decoded by fluorochrome conjugated oligonucleotides. Subsequently the same specimen was exposed to fluorescently labeled antibodies by cycles of antibody reaction, image acquisition and erasure of signal. Finally, specimens were stained with H&E to report on tissue integrity and correlate spatial orientation. The 2D image stacks were analyzed for transcript and antigen quantification and pattern recognition using both, pixel and segmented single-cell data (MACS iQ View Analysis Software). Across the specimens we identified more than 20 cell types including 12 immune cell subsets. The comparison of the tumor microenvironment, tumor-infiltrating immune cells (TILs) and their spatial organization in relation to the tumor cells allowed to identify differences and similarities across the tumor types. In summary we report here on a novel standardized and automated combined spatial RNA and protein expression analysis of the tumor microenvironment and tumor infiltrating immune cells across tumor types. The characterization dataset demonstrates the ability of our platform to perform standardized in-depth phenotyping of sample cohorts. This will enable discovery and further development of predictive and prognostic biomarkers critical to patient stratification for immunotherapy. Citation Format: Julia Femel, Emily Neil, Dongju Park, Fabio El Yassouri, Anijutta Appelshoffer, Erica Lloyd, Michael DiBuono, Henry Sauer, Hanna Lafayette, Hsinyi Smith, Jinling Wang, Dominic Mangiardi, Alex Makrigiorgos, Paurush Praveen, Silvia Rüberg, Werner Müller, Tanya Wantenaar, Robert Pinard, Andreas Bosio. Analysis of the immune microenvironment and tumor-infiltrating immune cells across different solid tumors by combined spatial transcriptomics and proteomics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5633.

PD02-04 SINGLE CELL TRANSCRIPTIONAL PROFILES OF BENIGN PROSTATIC HYPERPLASIA

You have accessJournal of UrologyCME1 Apr 2023PD02-04 SINGLE CELL TRANSCRIPTIONAL PROFILES OF BENIGN PROSTATIC HYPERPLASIA Rei Unno, Jon Akutagawa, Hanging Song, Heiko Yang, Thomas Chi, and Franklin W. Huang Rei UnnoRei Unno More articles by this author , Jon AkutagawaJon Akutagawa More articles by this author , Hanging SongHanging Song More articles by this author , Heiko YangHeiko Yang More articles by this author , Thomas ChiThomas Chi More articles by this author , and Franklin W. HuangFranklin W. Huang More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003219.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Benign prostatic hyperplasia (BPH) affects the majority of aging men and leads to lower urinary tract symptoms. Based on histological characteristics and bulk sequencing data, the pathophysiology and underlying mechanisms of BPH are poorly understood. The aim of this study was to investigate the transcriptional profile of BPH at single cell resolution to improve our understanding of the molecular features of BPH. METHODS: Fifteen BPH specimens were collected from patients undergoing holmium laser enucleation of the prostate. Cells from each specimen were isolated, captured, and sequenced using a bead-based single-cell RNA sequencing platform (Seq-well). Transcriptomic analysis of the gene expression matrices was performed using software including Scanpy and cellxgene. The absence of malignant pathologic diagnoses was independently confirmed. RESULTS: 16,234 cells from 15 BPH specimens were analyzed. By canonical cell type markers, we annotated three major cell types (epithelial, stromal, and immune cells). Using significantly upregulated genes in BPH compared to normal prostates previously derived from a bulk RNA sequencing comparison, the signature score of the bulk sequencing data was high only in the fibroblast populations. After performing a sub-clustering analysis of the 6249 epithelial cells, we identified four luminal cell subclusters showing similar levels of luminal epithelial markers KLK3, KLK2, and NKX3-1, as well as one distinct basal and one club cell population. Analysis of the differentially expressed genes among the four luminal subclusters revealed that luminal subcluster 4 was enriched in a specific signature gene set, including ribosomal genes and KLK4, which has been shown to be abundant in BPH, compared to the other subclusters. Gene ontology analysis using this gene set demonstrated that luminal subcluster 4 was associated with pathways such as oxidative phosphorylation and adipogenesis. Analyses of the BPH immune microenvironment showed a larger proportion of pro-inflammatory cells, such as M1 macrophages, compared to anti-inflammatory cells within the myeloid cell population. CONCLUSIONS: Single cell profiling of BPH identified epithelial cells, stromal cells, and immune cells. We confirmed the upregulation of previously derived BPH-associated genes in the fibroblast population and the enrichment of pro-inflammatory myeloid cells in the immune microenvironment. Within the epithelial cell population, we identified a luminal epithelial subcluster that may represent a cell state associated with BPH. Source of Funding: none © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e70 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Rei Unno More articles by this author Jon Akutagawa More articles by this author Hanging Song More articles by this author Heiko Yang More articles by this author Thomas Chi More articles by this author Franklin W. Huang More articles by this author Expand All Advertisement PDF downloadLoading ...

LBA03-09 CDH12 DRIVES THE DEVELOPMENT OF THE NEURONAL SUBTYPE IN MIBC

You have accessJournal of UrologyCME1 Apr 2023LBA03-09 CDH12 DRIVES THE DEVELOPMENT OF THE NEURONAL SUBTYPE IN MIBC Xingyu Chen, Mukta Basu, Simon Knott, and Dan Theodorescu Xingyu ChenXingyu Chen More articles by this author , Mukta BasuMukta Basu More articles by this author , Simon KnottSimon Knott More articles by this author , and Dan TheodorescuDan Theodorescu More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003426.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: We discovered a novel cell epithelial cell population we termed “C3” in muscle-invasive bladder cancer (MIBC) that is negatively prognostic for surgery and chemotherapy. This cell population shares similarities with the neuronal subtype MIBC (NEBC) which has limited treatment options and is driven by unclear molecular mechanisms. The objective of the study was to determine if NEBC is driven by some of the genes that are overexpressed in C3 cells. A top overexpressed and C3 cell specific gene that is also enriched in normal neuronal tissue is Cadherin 12 (N-Cadherin 2, CDH12). Hence, we initiated a study of this gene. METHODS: We overexpressed CDH12 in two murine MIBC cell lines (NA13 and MB49) and then inoculated them into mice. Tumor growth kinetics were studied, and single-nucleus RNA sequencing (snRNA-seq) and bulk RNA sequencing were then used to evaluate the tumors. Tissue samples from patients with MIBC were also analyzed using snRNA-seq and Co-detection by Indexing (CODEX) multiplexed immunofluorescence, and our bioinformatics analysis combined transcriptomic profiles from >2000 MIBC samples from published datasets, to characterize the transcriptional programs induced by enforcing CDH12 expression. RESULTS: Enforced expression of CDH12 led to enhanced tumor growth and the appearance of a neuroendocrine tumor phenotype. NEBC from human samples showed the highest expression of CDH12, and snRNA-seq results showed that NEBC also has the highest proportion of CDH12 epithelial cells and demonstrated co-expression of CDH12 and neuronal markers (NCAM1, CHGA, and SYP). RNA velocity analysis in murine and human tumors implicated CDH12 as a driver for the development of the neuronal subtype. CONCLUSIONS: To our knowledge, this study provides the first identification of a genetic driver leading to NEBC and implicates a novel gene, CDH12 in this process. Source of Funding: CA274375 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e1195 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Xingyu Chen More articles by this author Mukta Basu More articles by this author Simon Knott More articles by this author Dan Theodorescu More articles by this author Expand All Advertisement PDF downloadLoading ...

Cancer evolution as a new target of treatment paradigm

In clinical practice, the cancer treatment is certainly resulted from the biology of tumor growth, the modern understanding of which is based on the theory of genetic changes. In this regard, the effective strategy is the research and target on trigger mutations in the tumor. Obviously, a malignant neoplasm is the set of the different populations of epithelial, stromal and immune cells as well as intercellular matrix with dynamically changing blood flow, nutrients, acid-base state and oxygen concentration, which is almost not taken into account by clinicians.