Basal Markers Research Articles

Bronchial oncocytic carcinoma in an adult: a case report and literature review

BackgroundLung salivary-type tumors originating from bronchial submucosal glands are rare, only four types of salivary gland-type tumors are listed in 2015 WHO classification of lung tumors. Here, we report a rare case of oncocytic carcinoma (OC) in the right main bronchus.Case presentationA 34-year-old man presented to our hospital with a two-month history of recurrent hemoptysis and with one month of inspiratory dyspnea. Pulmonary function tests showed mild restrictive ventilatory dysfunction and severe diffusion dysfunction. Furthermore, the flow volume loop showed a variable extra-thoracic obstruction. Computed tomography (CT) of the chest revealed that a polypiform nodule of 13 mm in diameter was at the proximal right main bronchus. Testing for purified protein derivative was positive (category 2). The nodule was resected under bronchoscopy. The bronchial aspirate was negative for mycobacterium tuberculosis and tumor cells. The biopsy sample showed a solid and acinar predominant pattern with abundant eosinophilic cytoplasm. The bronchial mucosa was destroyed and replaced by tumor cells. The loose edematous stromal reaction could be seen in a local area. Immunohistochemically, tumor cells were positive for CK, EMA, Vimentin, CD117, CK7, S100, Mammaglobin and SOX10. Only scattered tumor cells were stained by basal cell markers, including CK5/6, P40 and P63. Electron microscopy revealed numerous swelling mitochondria with lacking mitochondrial cristae in tumor cells. Fluorescence in situ hybridization (FISH) testing for MAML2 and ETV6 rearrangement were negative. Next-generation sequencing analysis of 520 genes in the tissue biopsy specimen showed no somatic mutation. The diagnosis of OC was made. Subsequently, the patient underwent a right upper lobectomy with sleeve resection of the main bronchus and lymph dissection. No recurrent evidence was seen during two years of chest CT follow-up.ConclusionsTo our knowledge, this is the first case of primary OC in the bronchus. This patient has no recurrence during two years of follow-up, indicating that primary OC in the bronchus has the same favorable prognosis as in salivary glands. Moreover, complete excision and thorough sampling to know the invasive growth pattern is important to reach the correct diagnosis.

Characterization of 3D organotypic epithelial tissues reveals tonsil-specific differences in tonic interferon signaling.

Three-dimensional (3D) culturing techniques can recapitulate the stratified nature of multicellular epithelial tissues. Organotypic 3D epithelial tissue culture methods have several applications, including the study of tissue development and function, drug discovery and toxicity testing, host-pathogen interactions, and the development of tissue-engineered constructs for use in regenerative medicine. We grew 3D organotypic epithelial tissues from foreskin, cervix, and tonsil-derived primary cells and characterized the transcriptome of these in vitro tissue equivalents. Using the same 3D culturing method, all three tissues yielded stratified squamous epithelium, validated histologically using basal and superficial epithelial cell markers. The goal of this study was to use RNA-seq to compare gene expression patterns in these three types of epithelial tissues to gain a better understanding of the molecular mechanisms underlying their function and identify potential therapeutic targets for various diseases. Functional profiling by over-representation and gene set enrichment analysis revealed tissue-specific differences: i.e., cutaneous homeostasis and lipid metabolism in foreskin, extracellular matrix remodeling in cervix, and baseline innate immune differences in tonsil. Specifically, tonsillar epithelia may play an active role in shaping the immune microenvironment of the tonsil balancing inflammation and immune responses in the face of constant exposure to microbial insults. Overall, these data serve as a resource, with gene sets made available for the research community to explore, and as a foundation for understanding the epithelial heterogeneity and how it may impact their in vitro use. An online resource is available to investigate these data (https://viz.datascience.arizona.edu/3DEpiEx/).

Invasive urothelial carcinoma with chordoid features or abundant myxoid stroma: A reappraisal of morphologic spectrum and risk stratification based on molecular classification

Urothelial carcinoma (UC) with myxoid stroma or chordoid features is a rare diagnosis. We retrospectively collected data from 17 cases of diagnosed UC with myxoid stroma, mucin production, or chordoid features. We aimed to investigate the molecular subtypes of this neoplasm and to assess subtype correlations with clinical outcomes. Immunohistochemical (IHC) staining with a panel composed of markers for basal subtypes (CK5/6, CK14, and CD44) and luminal subtypes (GATA3, FOXA1, and CK20) was performed. Morphologically, all cases included an at least partial conventional UC component, with the first histologic pattern, named as “typical”, characterized by a small- or medium-sized tumor cell nest. The second histologic pattern, named as “chordoid”, was characterized by tumor cells with cording that mimic extra-skeletal myxoid chondrosarcoma or chordoma, and the third histologic pattern, named as “sarcomatoid”, was characterized by non-cohesive spindle tumor cells with a mucin-producing or myxoid stroma background. The “typical” cases showed [CK5/6- CK14- CD44-] [GATA3 + FOXA1 + CK20-] IHC results and was classified as lumina subtype. The “chordoid” cases showed [CK5/6 + CK14 + CD44-] [GATA3- FOXA1- CK20-] IHC results and was classified as basal subtype, and the “sarcomatoid” cases showed [CK5/6- CK14- CD44+] [GATA3- FOXA1- CK20-] IHC results and was “not classified”. All pT3 cases and all cases with lymph node (LN) metastasis belonged to the “sarcomatoid” pattern. All patients who had metastasis or died showed the “chordoid” or “sarcomatoid” morphology. Our findings suggest that UC with myxoid stroma/chordoid features shows characteristic expression of luminal and basal markers and different prognosis according to the morphologic pattern spectrum.

Cigarette smoke-induced injury induces distinct sex-specific transcriptional signatures in mice tracheal epithelial cells.

The airway epithelial barrier is crucial for defending against respiratory insults and diseases. Disruption of epithelial integrity contributes to respiratory diseases, and sex-specific differences in susceptibility and severity have been observed. However, sex-specific differences in the context of respiratory diseases are often overlooked, especially in murine models. In this study, we investigated the in vitro transcriptomics of male and female murine tracheal epithelial cells (mTECs) in response to chronic cigarette smoke (CS) exposure using an International Organization for Standardization (ISO) puff regimen. Our findings reveal sex-specific differences in the baseline characteristics of airway epithelial cells. Female mTECs demonstrated stronger barrier function and higher ciliary function compared with males. The barrier function was disrupted in both males and females following chronic CS, but the difference was more significant in females due to their higher baseline. Female mice exhibited transcriptional signatures suggesting dedifferentiation with increased basal cells and markers of cellular senescence. Pathway analysis indicated potential protective roles of planar cell polarity (PCP) in preventing dedifferentiation in male mice exposed to CS. We also observed sex-specific differences in the DNA damage response and antioxidant levels, suggesting distinct mechanisms underlying cellular stress. Understanding these sex-specific mechanisms could facilitate the development of targeted therapeutic strategies for lung diseases associated with environmental insults. Recognizing sex-based differences in disease susceptibility and treatment response can lead to personalized care and improved outcomes. Clinical trials should consider sex as a biological variable to develop effective interventions that address the unique differences between men and women in respiratory diseases.NEW & NOTEWORTHY The study underscores the importance of considering sex-specific differences in the airway epithelium in respiratory diseases such as COPD. Differences in gene expression between males and females at baseline and in response to chronic injury in the airway epithelium could have implications on disease susceptibility, both in COPD and other respiratory diseases. Therefore, understanding these differences is crucial for developing targeted therapies to treat respiratory diseases based on a sex-specific manner.

Effects of an Adipose Mesenchymal Stem Cell-Derived Conditioned medium and TGF-β1 on Human Keratinocytes In Vitro.

Human keratinocytes play a crucial role during skin wound healing and in skin replacement therapies. The secretome of adipose-derived stem cells (ASCs) has been shown to secrete pro-healing factors, among which include TGF-β1, which is essential for keratinocyte migration and the re-epithelialization of cutaneous wounds during skin wound healing. The benefits of an ASC conditioned medium (ASC-CM) are primarily orchestrated by trophic factors that mediate autocrine and paracrine effects in keratinocytes. Here, we evaluated the composition and the innate characteristics of the ASC secretome and its biological effects on keratinocyte maturation and wound healing in vitro. In particular, we detected high levels of different growth factors, such as HGF, FGFb, and VEGF, and other factors, such as TIMP1 and 4, IL8, PAI-1, uPA, and IGFBP-3, in the ASC-CM. Further, we investigated, using immunofluorescence and flow cytometry, the distinct effects of a human ASC-CM and/or synthetic TGF-β1 on human keratinocyte proliferation, migration, and cell apoptosis suppression. We demonstrated that the ASC-CM increased keratinocyte proliferation as compared to TGF-β1 treatment. Further, we found that the ASC-CM exerted cell cycle progression in keratinocytes via regulating the phases G1, S, and G2/M. In particular, cells subjected to the ASC-CM demonstrated increased DNA synthesis (S phase) compared to the TGF-β1-treated KCs, which showed a pronounced G0/G1 phase. Furthermore, both the ASC-CM and TGF-β1 conditions resulted in a decreased expression of the late differentiation marker CK10 in human keratinocytes in vitro, whereas both treatments enhanced transglutaminase 3 and loricrin expression. Interestingly, the ASC-CM promoted significantly increased numbers of keratinocytes expressing epidermal basal keratinocyte markers, such DLL1 and Jagged2 Notch ligands, whereas those ligands were significantly decreased in TGF-β1-treated keratinocytes. In conclusion, our findings suggest that the ASC-CM is a potent stimulator of human keratinocyte proliferation in vitro, particularly supporting basal keratinocytes, which are crucial for a successful skin coverage after transplantation. In contrast, TGF-β1 treatment decreased keratinocyte proliferation and specifically increased the expression of differentiation markers in vitro.

Effects of Heparan sulfate acetyl-CoA: Alpha-glucosaminide N-acetyltransferase (HGSNAT) inactivation on the structure and function of epithelial and immune cells of the testis and epididymis and sperm parameters in adult mice.

Heparan sulfate (HS), an abundant component of the apical cell surface and basement membrane, belongs to the glycosaminoglycan family of carbohydrates covalently linked to proteins called heparan sulfate proteoglycans. After endocytosis, HS is degraded in the lysosome by several enzymes, including heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT), and in its absence causes Mucopolysaccharidosis III type C (Sanfilippo type C). Since endocytosis occurs in epithelial cells of the testis and epididymis, we examined the morphological effects of Hgsnat inactivation in these organs. In the testis, Hgsnat knockout (Hgsnat-Geo) mice revealed statistically significant decrease in tubule and epithelial profile area of seminiferous tubules. Electron microscopy (EM) analysis revealed cross-sectional tubule profiles with normal and moderately to severely altered appearances. Abnormalities in Sertoli cells and blood-testis barrier and the absence of germ cells in some tubules were noted along with altered morphology of sperm, sperm motility parameters and a reduction in fertilization rates in vitro. Along with quantitatively increased epithelial and tubular profile areas in the epididymis, EM demonstrated significant accumulations of electrolucent lysosomes in the caput-cauda regions that were reactive for cathepsin D and prosaposin antibodies. Lysosomes with similar storage materials were also found in basal, clear and myoid cells. In the mid/basal region of the epithelium of caput-cauda regions of KO mice, large vacuolated cells, unreactive for cytokeratin 5, a basal cell marker, were identified morphologically as epididymal mononuclear phagocytes (eMPs). The cytoplasm of the eMPs was occupied by a gigantic lysosome suggesting an active role of these cells in removing debris from the epithelium. Some eMPs were found in proximity to T-lymphocytes, a feature of dendritic cells. Taken together, our results reveal that upon Hgsnat inactivation, morphological alterations occur to the testis affecting sperm morphology and motility parameters and abnormal lysosomes in epididymal epithelial cells, indicative of a lysosomal storage disease.

Tertiary lymphoid structure and neutrophil-lymphocyte ratio coordinately predict outcome of pembrolizumab.

Emerging evidence suggests that the presence of tertiary lymphoid structures (TLS) and neutrophil-lymphocyte ratio (NLR) in peripheral blood is associated with the treatment response to checkpoint inhibitors (CPIs), whereas there is limited knowledge regarding whether these factors reciprocally impact the treatment outcomes of CPIs in metastatic urothelial carcinoma (mUC). Herein, we investigated treatment outcomes of platinum-refractory mUC patients (50 cases with whole-exome and transcriptome sequencing) treated with pembrolizumab. The pathological review identified 24% of cases of TLS in the specimens. There was no significant difference in the NLR between the TLS- and TLS+ groups (p = 0.153). In the lower NLR group, both overall survival and progression-free survival were significantly longer in patients with TLS than in those without TLS, whereas the favorable outcomes associated with TLS were not observed in patients in the higher NLR group. We explored transcriptomic differences in UC with TLS. The TLS was comparably observed between luminal (20%) and basal (25%) tumor subtypes (p = 0.736). Exploring putative immune-checkpoint genes revealed that ICOSLG (B7-H2) was significantly increased in tumors with lower NLR. KRT expression levels exhibited higher basal cell markers (KRT5 and KRT17) in the higher NLR group and lower differentiated cell markers (KRT8 and KRT18) in patients with TLS. In conclusion, the improved outcomes of pembrolizumab treatment in mUC are restricted to patients with lower NLR. Our findings begin to elucidate a distinct molecular pattern for the presence of TLS according to the NLR in peripheral blood.

Epithelial polarity-driven membrane separation but not cavitation regulates lumen formation of rat eccrine sweat glands

BackgroundEach eccrine sweat gland (ESG) is a single-tubular structure with a central lumen, and the formation of hollow lumen in the initial solid cell mass is a key developmental process. To date, there are no reports on the mechanism of native ESG lumen formation. MethodsTo investigate the lumen morphogenesis and the lumen formation mechanisms of Sprague-Dawley (SD) rat ESGs, SD rat hind-footpads at E20.5, P1–P5, P7, P9, P12, P21, P28 and P56 were obtained. The lumen morphogenesis of ESGs was examined by HE staining and immunofluorescence staining for polarity markers. The possible mechanisms of lumen formation were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay and autophagy marker LC3B immunofluorescence staining, and further explored by ouabain intervention experiment. ResultsIn SD rat ESGs, the microlumen was formed at P1, and the small intact lumen with apical-basal polarity appeared at P3. The expression of apical marker F-actin, basal marker Laminin, basolateral marker E-cadherin was consistent with the timing of lumen formation of SD rat ESGs. During rat ESG development, apoptosis and autophagy were not detected. However, inhibition of Na+-K+-ATPase (NKA) with ouabain resulted in decreased lumen size, although neither the timing of lumen formation nor the expression of polarity proteins was altered. ConclusionsEpithelial polarity-driven membrane separation but not cavitation regulates lumen formation of SD rat ESGs. NKA-regulated fluid accumulation drives lumen expansion.

Depressed, stressed, and inflamed: C-reactive protein linked with depression symptoms in midlife women with both childhood and current life stress.

To determine whether the relationship between inflammatory factors and clinically significant depression symptoms is moderated by high exposure to adverse childhood experiences and current life stressors in a longitudinal community cohort of midlife women. Methods: Participants from the Penn Ovarian Ageing Study community cohort (age at baseline: M=45.3 [SD=3.8]) were included in analyses if they had a blood sample measuring basal inflammatory markers during at least one visit where depression symptom severity and current stressful life events were also assessed (N=142, average number of visits per participant=1.75 [SD=0.92]). Approximately annually over the course of 16years, participants self-reported depression symptom severity using the Centre for Epidemiologic Studies Depression (CESD) Scale, provided menstrual diaries to determine menopause stage, and contributed blood samples. Residual blood samples were assayed for interleukin (IL)-6, IL 1-beta (IL-1β), tumour necrosis factor alpha (TNF-α), and high sensitivity C-reactive protein (hsCRP). Early life stress was quantified using the Adverse Childhood Experiences questionnaire (low [0-1 experience(s)] versus high [≥ 2 experiences]). Current stressful life events were assessed using a structured interview (low [0-1 events] vs. high [≥ 2 events]). Generalised estimating equation models were used to model associations with the outcome of interest-clinically significant depression symptoms (CESD ≥16)-and risk factors: inflammatory marker levels (log transformed), adverse childhood experiences group, and current life stressors group. Covariates included menopause stage, age at study baseline, body mass index, race, and smoking status. We found a significant three-way interaction between log hsCRP levels, adverse childhood experiences group, and current life stressors group on likelihood of experiencing clinically significant depression symptoms (OR: 4.33; 95% CI: 1.22, 15.46; p=0.024) after adjusting for covariates. Solely for women with high adverse childhood experiences and with high current life stressors, higher hsCRP was associated with higher odds of having clinically significant depression symptoms (OR: 1.46; 95% CI 1.07, 1.98; p=0.016). This three-way interaction was not significant for IL-6, IL-1β, or TNF-α. For women in midlife with exposure to high adverse childhood experiences and multiple current life stressors, elevated levels of CRP were uniquely associated with clinically significant depression symptoms. Early life adversity and current life stressors represent identifiable individual risk factors whose negative impact may be curtailed with inventions to target inflammation in midlife women.

Morphology, immunohistochemistry characteristics, and clinical presentation of microcystic urothelial carcinoma: a series of 10 cases

BackgroundMicrocystic urothelial carcinoma (MUC) is a rare variant of urothelial carcinoma with histological appearances similar to begin lesions. Thus far, approximately 50 cases have been reported. Here, we investigated the clinicopathological features of MUC.MethodsClinical data and paraffin-embedded tissue blocks were collected. Immunohistochemical staining and polymerase chain reaction–Sanger sequencing were performed to detect the phenotype and TERT mutation status of MUC, respectively.ResultsThe mean patient age was 58.8 ± 14.5 years, with a male predominance (8:2). The pathological stage was T1 in one case, T2 in three cases, T3 in four cases, and T4 in two cases. Tumor metastases or death occurred in all five patients who were followed up within 1–3 years. Histological analyses revealed microcystic, tubular, cribriform, and occasionally cord-like structures, which generally lacked interstitial reactions. The lumens were empty, contained eosinophilic secretion, or were filled with mucin. The microcysts/tubules/cribriform patterns were lined by flat, cuboid, signet ring, or columnar types of epithelia. The cuboid, signet ring, and columnar types represented “glandular metaplasia” or glandular differentiation of urothelial carcinoma. Immunohistochemistry analyses revealed distinct co-expression patterns involving the luminal markers FOXA1 and GATA3, as well as the basal markers CK5/6 and CD44. All 10 cases exhibited a luminal phenotype according to the GATA3+/CK14- criterion, whereas nine cases exhibited a luminal phenotype according to the FOXA1+/CK14- criterion. The telomerase reverse transcriptase-C228T mutation was detected in seven cases.ConclusionsMUC is a rare variant with a deceptively benign form of urothelial carcinoma, which is generally identified as a late-stage tumor with a poor prognosis. It exhibits distinct co-expression of luminal and basal markers, along with the TERT-C228T mutation.

The koala (Phascolarctos cinereus) prostate: a comprehensive histological and immunohistochemical investigation.

The prostate of the koala (Phascolarctos cinereus), and of marsupials more generally, is the primary contributor of seminal fluid, yet comparatively little is known about its microanatomy or biochemistry. This study explored evidence of parenchymal segmentation of the koala prostate. The prostate of three sexually mature koalas were processed for histopathology, histochemistry (Masson's trichrome, Alcian Blue, periodic acid Schiff staining), and immunohistochemistry using basal (tumor protein 63, cytokeratin 14) and luminal (cytokeratin 8/18, prostate specific antigen, androgen receptor) markers. Results confirmed clear segmentation of the koala prostate into three zones, anterior, central, and posterior, characterized by differences in the proportion of glandular tissue, as well as the thickness of collagen fibers; there were also distinct differences in the secretions produced in each zone. Based on immunohistochemistry, the koala prostate showed evidence of both basal proliferative and luminal secretory cells. The ratio of cell types varied across the three segments, with the central segment housing the highest density of basal cells. Globular bodies produced in the anterior zone were shown to possess the same markers as those described for human prostasomes. This study is the first to comprehensively document the marsupial prostate in terms of microanatomy and corresponding immunohistochemistry. While further biochemical analysis, such as proteomics of each segment will better define the relative functions of each tissue, the data presented here are consistent with the hypothesis that the koala prostate potentially represents an example of an ontological stage in the evolutionary differentiation of male eutherian accessory glands.

Estrogen receptor alpha regulates uterine epithelial lineage specification and homeostasis

Postnatal development of the uterus involves specification of undifferentiated epithelium into uterine-type epithelium. That specification is regulated by stromal-epithelial interactions as well as intrinsic cell-specific transcription factors and gene regulatory networks. This study utilized mouse genetic models of Esr1 deletion, endometrial epithelial organoids (EEO), and organoid-stromal co-cultures to decipher the role of Esr1 in uterine epithelial development. Organoids derived from wild-type (WT) mice developed a normal single layer of columnar epithelium. In contrast, EEO from Esr1 null mice developed a multilayered stratified squamous type of epithelium with basal cells. Co-culturing Esr1 null epithelium with WT uterine stromal fibroblasts inhibited basal cell development. Of note, estrogen treatment of EEO-stromal co-cultures and Esr1 conditional knockout mice increased basal epithelial cell markers. Collectively, these findings suggest that Esr1 regulates uterine epithelium lineage plasticity and homeostasis and loss of ESR1 promotes altered luminal-to-basal differentiation driven by ESR1-mediated paracrine factors from the stroma.

Urothelium-Specific Expression of Mutationally Activated Pik3ca Initiates Early Lesions of Noninvasive Bladder Cancer

Although approximately 70% of bladder cancers are noninvasive and have high recurrence rates, early-stage disease is understudied. The lack of models to validate the contribution of molecular drivers of bladder tumorigenesis is a significant issue. Although mutations in PIK3CA are frequent in human bladder cancer, an invivo model for understanding their contribution to bladder tumorigenesis is unavailable. Therefore, a Upk2-Cre/Pik3caH1047R mouse model expressing one or two R26-Pik3caH1047R alleles in a urothelium-specific manner was generated. Pik3caH1047R functionality was confirmed by quantifying Akt phosphorylation, and mice were characterized by assessing urothelial thickness, nuclear atypia, and expression of luminal and basal markers at 6 and 12 months of age. While at 6 months, Pik3caH1047R mice developed increased urothelial thickness and nuclear atypia, progressive disease was not observed at 12 months. Immunohistochemistry showed urothelium maintained luminal differentiation characterized by high forkhead box A1 (Foxa1) and peroxisome proliferator-activated receptor γ expression. Surprisingly, Pik3caH1047R mice subjected to low-dose carcinogen exposure [N-butyl-N-(4-hydroxybutyl)nitrosamine] exhibited no significant differences after exposure relative to mice without exposure. Furthermore, single-sample gene set enrichment analysis of invasive human tumors showed those with mutant PIK3CA did not exhibit significantly increased phosphatidylinositol 3-kinase/AKT pathway activity compared with wild-type PIK3CA tumors. Overall, these data suggest that Pik3caH1047R can elicit early tumorigenic changes in the urothelium, but progression to invasion may require additional genetic alterations.

Beneficial Short-Term Effects of Bariatric Surgery on Nutritional Inflammatory Profile and Metabolic Biomarkers.

Bariatric surgery (BS) has several potential metabolic benefits. However, little is known about its impact on changes in the inflammatory potential of diet and its effect on inflammatory and metabolic markers. This study aimed to assess the short-term beneficial effects of BS on dietary inflammatory potential and inflammatory and metabolic markers. Participants (n = 20) were evaluated 3 months before and after BS. Body mass, body mass index, anthropometric measurements, fat mass, fat-free mass, visceral fat, skeletal muscle mass, basal metabolic rate, serum lipids, HOMA-IR, QUICKI and inflammatory markers, including leptin, adiponectin, adiponectin/leptin ratio and plasminogen activator inhibitor-1 (PAI-1), were evaluated. Diet data were collected using a 3-day diet record and the dietary inflammatory index (DII®) and energy-adjusted dietary inflammatory index (E-DIITM) scores were computed. There was a reduction in DII® (2.56 vs 2.13) and E-DIITM (2.18 vs 0.45) indicating an improvement in inflammatory nutritional profile. Moreover, there were increases in the adiponectin/leptin ratio (0.08 vs 0.21) and QUICKI scores (0.31 vs 0.37), and reductions in leptin (36.66 vs 11.41 ng/ml) and HOMA-IR scores (3.93 vs 1.50). There were also improvements in body composition and anthropometric parameters. BS promotes changes in metabolic profile, inflammatory state and food intake and these modifications appeared to be associated with improvements in diet-related inflammation, an increase in the adiponectin/leptin ratio and a reduction in leptin. These results contribute to knowledge on the contribution bariatric surgery can make to the treatment of obesity and the reduction of related comorbidities.

Abstract PR007: ERG-driven luminal prostate cancers emerge from Ck5+/Nkx3.1+ basal cells

Abstract TMPRSS2-ERG translocations are initiating lesions in nearly 50% of human prostate adenocarcinomas, often in conjunction with PTEN loss. In mice, prostate-specific expression of ERG using a Probasin-Cre driver (Pb-Cre4) is sufficient to induce invasive Ck8+ luminal adenocarcinoma with high penetrance in the context of Pten loss. Because TMPRSS2 expression in the human prostate is androgen-regulated and thereby largely restricted to luminal epithelial cells, it has been assumed that luminal cells are the likely cell of origin for TMPRSS2-ERG positive prostate cancers. To gain further support for this hypothesis, we examined the early stages of luminal adenocarcinoma initiation in the Pb-Cre ERG/Pten model at a single cell level. To our surprise, we found a highly proliferative population of ERG+ Ck5+ (a putative basal marker) cells that are enriched at the invasive front in a background of more canonical ERG+/Ck8+/Ck5- luminal tumor cells. We next performed lineage tracing experiments using Ck5-, Ck8- as well as Nkx3.1-Cre drivers, introduced via viral injection or tamoxifen induction, which collectively support the hypothesis that ERG-driven tumors originate in a subset of basal cells defined by Ck5+/Nkx3.1+. This conclusion is further supported by orthotopic transplantation of freshly isolated primary mouse prostate basal (Cd49f high) or luminal (Cd24 high) cells showing that ERG+/Ck8+ tumors arise preferentially from basal cells following ex vivo ERG/Pten activation. Time course experiments in organoids and in vivo show that ERG promotes Ck8+ luminal differentiation from highly proliferative Ck5+ cells, progressing through a Ck5+/Ck8+ intermediate cell state. Collectively, these data implicate a tumor initiating cell population defined by a mix of basal (Ck5) and luminal (Nkx3.1) markers that, under the influence of ERG expression, can expand and invade while retaining full luminal differentiation potential. Our results also suggest that widely held assumptions regarding luminal-specific expression of canonical markers such as Nkx3.1 and Probasin may need revision. Citation Format: Weiran Feng, Erik Ladewig, Nazifa Salsabeel, Huiyong Zhao, Young Sun Lee, Anuradha Gopalan, Hanzhi Luo, Wenfei Kang, Ning Fan, Eric Rosiek, Ignas Masilionis, Katia Manova-Todorova, Ronan Chaligné, Elisa de Stanchina, Brett S. Carver, Yu Chen, Dong Gao, Christina S. Leslie, Charles L. Sawyers. ERG-driven luminal prostate cancers emerge from Ck5+/Nkx3.1+ basal cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr PR007.

Abstract A044: Generation of a new mouse model to study the role of oncofetal Cripto in aggressive lethal prostate cancer

Abstract Introduction: Early stages of prostate cancer (PCa) are commonly treated with surgery and androgen-deprivation therapy, but PCa can turn castration resistant due to pre-existing stem cell-like cells which might re-initiate tumor growth and lead to metastasis. Previously, it has been shown that human tumors express high levels of Cripto, an oncofetal protein, for this reason our hypothesis is that Cripto might play a role in PCa initiation and progression. We aim to study its oncogenic features in GEMMs models that are representative of early and late metastatic PCa. Methods: We performed conditional Cripto (CRIPTOflox/flox) knock out on N (Nkx3.1CreERT2, R26 LSL-YFP/LSL-YFP), NP (Nkx3.1CreERT2; Ptenflox/flox, R26 LSL-YFP/LSL-YFP) and NPK (Nkx3.1CreERT2; Ptenflox/flox; KrasLSL-G12D/+, R26 LSL-YFP/LSL-YFP) to generate respectively NC, NPC and NPKC. N animals mirror normal epithelium, whereas first stage of the disease, high-grade prostatic intraepithelial neoplasia (PIN)/carcinoma lesions with local invasive epithelium, are seen in NP. More advanced stage with invasive prostate adenocarcinoma with metastasis are developed in NPK. In vivo experiments’ workflow was the following: 8-weeks old mice were castrated and induced one month later with 5 daily injections of tamoxifen, after 2.5 weeks, animals were weekly treated with testosterone (10 weeks). Organoids were generated from YFP+/− single cell population recovered by FACS sorting. Results: Histopathological evaluation of newly generated NPC and NPKC showed presence of mPIN (100%, nNPC= 6, nNPKC=2), with a dominant cribriform morphology. NPKC additionally, features invasive PCa with an extent dominant pattern of 100%, showing portions with dense stroma forming whorl-like structures and occasional sheet-like accumulations of polygonal in stroma of regions with mPIN. In general, NPC and NPKC feature a more reactive stroma with a mild/moderate inflammation compared to the published models. OrganoidsYFP+ recapitulate molecular prostate tissue features, expressing luminal and basal markers. Organoids’ morphology varies consistently: N and NC are low in density and present a more cystic morphology, which is consistent with low-grade PIN phenotypes, whereas NP and NPK organoids are solid and denser which mimics an oncogenic transformation. In general, NC, NPC and NPKC organoids present a higher percentage of hollow organoids compared to N, NP and NPK respectively. Conclusions: NPC and NPKC phenotype specifically, showed important stromal alterations suggesting that Cripto might play a role not only on the epithelial compartment as well as in the stroma. For this reason, studies on secreted Cripto inhibition with ALK4-Fc are on-going. Our results show that organoids are an efficient in vitro model replicating different phenotypes seen in vivo. Citation Format: Elisa Rodrigues Sousa, Eugenio Zoni, Mario Scarpa, Marta De Menna, Allen Abey Alexander, Simone De Brot, George N. Thalmann, Marianna Kruithof-de Julio. Generation of a new mouse model to study the role of oncofetal Cripto in aggressive lethal prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A044.

Abstract B006: Identifying the molecular signature of bi-potent prostate epithelial cells through scRNA-seq

Abstract Introduction: Recent studies have shown that cells undergoing oncogenic processes usurp normal developmental pathways to escape regulators of unsolicited growth. Basal to luminal differentiation in the prostate serves as another cellular process that can go awry, the dysregulation of which can enable oncogenesis in the prostate. Studies have found bi-potent cells in prostate epithelium and speculate those to be cells of origin in prostate cancer. Methods and Results: Through single-cell RNA sequencing we investigated basal to luminal differentiation of normal human prostate epithelial cells across timepoints Days 0, 4, 8, 12, 16, and 20 by stimulating with FGF-7 (20ng/mL) and R1881 (10nM) and explored the data to identify unique populations present in the differentiation process. Our results show spatial separation at various timepoints with distinct transcriptomics profile at Day 0, Day 16, and Day 20. As expected, populations present at Day 0 are also present across all timepoint, though enrichment of various distinct populations emerged throughout the differentiation process. Using trajectory analysis (Monocle3) and the Cell Surface Protein Atlas, we profiled surface proteins for each cluster at Day 0 and identified a set of unique surface markers (SLC3A, LYPD3, TACSTD2, DSC2) from undifferentiated basal cells that express both basal (TP63+, K14+) and luminal markers (K8+, K18+), potentially serving as a bi-potent prostate epithelial cells. Conclusion: Our data have identified unique populations present in our prostate differentiation model expressing both luminal and basal signatures which can be isolated using identified surface markers to investigate the role of bi-potent cells in prostate differentiation and oncogenesis. Citation Format: Hunain Khawaja, Cynthia Miranti. Identifying the molecular signature of bi-potent prostate epithelial cells through scRNA-seq [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B006.

Abstract A085: A luminal intermediate cell state maintains long-term prostate homeostasis and contributes to tumorigenesis

Abstract Cellular heterogeneity poses tremendous challenges for developing cell-targeted therapies and biomarkers of clinically significant prostate cancer. The origins of this heterogeneity within the cellular complexities of normal adult and aging tissue remain unknown leaving important cellular states and transcriptional programs that allow the expansion of malignant clones in early cancer unidentified. These unresolved questions hamper the development of novel therapies designed to block the deregulated proliferation of prostate epithelial cells. To define cell states that contribute to early cancer development, we performed in vivo lineage tracing with multicolor reporters, whole organ mapping and single cell transcriptomics of normal and malignant prostate from genetically-engineered mouse models. We show here that the long-term luminal homeostasis is maintained by multiple clones with various fitness levels. Long-term clonal expansions are enabled by a luminal transcriptional state harboring basal markers (Luminal Intermediate, LumI) and controlled by Wnt/p63 signaling. Moreover, LumI cells greatly expand during early stages of tumorigenesis in several mouse models of prostate cancer. Specific genetic ablation of p63 from luminal cells in vivo decreases clonal activity in homeostasis and reduces the formation of aggressive clones in prostate tumor models. Finally, the LumI cells and Wnt signaling appear to significantly increase in human aging prostate and prostate cancer samples, highlighting the importance of this hybrid cell state for human pathologies with potential translational impact. Our models reveal a continuity of this cell state from normal homeostasis to aging and early cancer with specific alterations at each stage. Understanding how the luminal intermediate cell state is regulated in homeostasis and deregulated in hyperproliferative contexts is crucial for the development of prognostic markers and new therapeutic interventions for prostate diseases. Citation Format: Fu Luo, Lara F. Tshering, Karis Tutuska, Mariola Szenk, Diana Rubel, James G. Rail, Savanah Russ, Jingxuan Liu, Alice Nemajerova, Gábor Balázsi, Flaminia Talos. A luminal intermediate cell state maintains long-term prostate homeostasis and contributes to tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A085.

Transcriptomic landscape of early hair follicle and epidermal development

Morphogenesis of ectodermal organs, such as hair, tooth, and mammary gland, starts with the formation of local epithelial thickenings, or placodes, but it remains to be determined how distinct cell types and differentiation programs are established during ontogeny. Here, we use bulk and single-cell transcriptomics andpseudotime modeling to address these questions in developing hair follicles and epidermis and produce a comprehensive transcriptomic profile of cellular populations in the hair placode and interplacodal epithelium. We report previously unknown cell populations and marker genes, including early suprabasal and genuine interfollicular basal markers, and propose the identity of suprabasal progenitors. By uncovering four different hair placode cell populations organized in three spatially distinct areas, with fine gene expression gradients between them, we posit early biases in cell fate establishment. This work is accompanied by a readily accessible online tool to stimulate further research on skin appendages and their progenitors.

Enhanced detection and characterization of targetable EGFR and ERBB2 exon20 insertion mutations in urothelial carcinoma.

e16500 Background: EGFR and ERBB2 exon 20 insertion (exon20ins) driver mutations are well described in non-small cell lung cancer due to their resistance to early generation EGFR tyrosine kinase inhibitors. This has led to the development and ultimate approval of exon20ins specific inhibitors, such as mobocertinib. Previous studies have reported EGFR and ERBB2 exon20ins mutations in 0.15%-1.91% of urothelial carcinoma (UC) patients, but this is likely an underrepresentation as indel variants are often under detected by commonly used NGS-variant analysis tools. Additionally, little is known about the biological consequences of EGFR and ERBB2 exon20ins mutations in UC. In this study, we aimed to: 1) characterize the prevalence of EGFR and ERBB2 exon20ins mutations in UC; 2) assess how EGFR and ERBB2 exon20ins mutations impact malignant transformation; and 3) evaluate the sensitivity of UC cell lines with the exon20ins to mobocertinib. Methods: UC patient datasets were realigned using the indel caller, ABRA2, then EGFR and ERBB2 exon20ins frequencies were calculated. Next, parental KT1970 urothelial cells expressing either EGFR WT, ERBB2 WT, or exon20ins mutation constructs (EGFRinsH [insH] or ERBB2insYVMA [YVMA]) were generated and in vitro transformation assays were performed. Anchorage-independent growth was assessed with soft agar assay. Engineered urothelial organoids with various exon20ins variants were created to assess for their ability to differentiate. Finally, sensitivity to erlotinib and mobocertinib was determined. Results: ABRA2 identified a prevalence of ERBB2 and EGFR Exon20ins mutations in 2.5% (7/275) of urothelial tumors in the IMVigor210 patient cohort. ERBB2 insYVMA expressing organoids had the highest basal marker (KRT5) expression in proliferation media which decreased in differentiation media. In the soft agar assay, YVMA cells had a 9.26 fold increase in colony formation (CF) compared to the ERBB2 WT counterpart and insH had a 6.02 fold increase in CF over the EGFR WT counterpart. The IC50 for mobocertinib (M) was significantly lower than erlotinib (E) in the exon20ins cell lines (insH M: 0.123 uM, E:10.69 uM / YVMA M: 0.57 uM, E: 13.54 uM). The difference in IC50 values for mobocertinib vs erlotinib in the WT cells was less pronounced. Conclusions: The prevalence of EFGR and ERBB2 exon20 insertion mutations is likely unrepresented in urothelial carcinoma by current variant calling pipelines. The ERBB2 YVMA mutation showed a more aggressive phenotype and possible basal pattern in vitro which may have implications in terms of prognosis and treatment considerations. Both EGFR and ERBB2 exon20ins urothelial cells were more sensitive to mobocertinib. This study provides evidence that exon20ins specific inhibitors could offer additional targeted therapy options for patients with urothelial cancer who have EGFRinsH and ERBB2insYVMA mutations.