Basal-like Cells Research Articles

Abstract B010: Investigating endoplasmic reticulum stress responses in pancreatic ductal adenocarcinoma under lipid imbalance

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related death with a five-year survival rate of 10%. The complex genetic makeup and heterogeneity of its microenvironment make PDAC difficult to treat. Desmoplasia, a major feature of PDAC’s TME, are dense fibrotic and inflammatory stromal regions. Desmoplastic reaction results in poor intratumoral perfusion and elevated interstitial fluid pressure, creating regions with severe oxygen and nutrient deprivation. One result is a deficiency in unsaturated fatty acids (uFAs) because the de novo synthesis of uFAs requires oxygen. Our results indicate that PDAC cells experience endoplasmic reticulum (ER) stress and trigger subsequent ER stress responses under uFA limited conditions in vitro. The ER stress responses are governed by three major stress sensors: Inositol-requiring enzyme 1𝛼 (IRE1𝛼), PKR-like ER kinase (PERK) and activating transcription factor 6𝛼 (ATF6𝛼). We hypothesize that ER stress responses are tumor promoting in PDAC and used as a survival strategy under uFA deprivation. In addition, previous studies showed that MYC-driven cancer cells are highly reliant on the ER stress response pathways. Therefore, we hypothesize that the two major PDAC transcriptional subtypes “Classical” and “Basal-like” have distinct dependencies on ER stress response pathways, with the basal-like subtype being more dependent due to an enrichment of MYC gene signatures. Our results suggest that opposite to cancer cell promoting, ER stress responses mediated by IRE1𝛼, PERK and ATF6𝛼 are cytotoxic in vitro under uFA deprived conditions. By analyzing the published bulk RNA-seq from patient samples, we found that the basal-like PDAC subtype is enriched with an ER stress gene signature. To avoid contamination from other cell populations, we are currently analyzing a published scRNA-seq dataset from 15 independent patient samples. We will investigate whether the ER stress signature is also enhanced in basal-like cells. Basal-like PDAC cells are usually more mesenchymal; we therefore created isogenic “epithelial” and “mesenchymal” lines to mimic PDAC subtypes. However, we did not see signaling preferences between the lines. One caveat is that the basal-like subtype have many more features than being mesenchymal, and our isogenic system might not be a faithful modeling of the subtypes. We are studying whether hypoxic PDAC cells experience ER stress in vivo due to a deprivation in uFAs using multiplex fluorescent imaging, stimulated Raman microscopy and/or mass spectrometry. We are evaluating the role of ER stress responses in vivo and see whether inhibiting ER stress response alone or in combination with other reagents gives rise to potential therapeutic opportunities. This project will provide a better understanding of the role ER stress responses play under uFA deprivation and will also enable us to further understand PDAC subtypes in terms of how they utilize ER stress responses. Citation Format: Yanqing (Christine) Jiang, Xu Han, Mai Wang, Raymond Ng, Pamela Burgess-Jones, John Tobias, Nathan Coffey, Andrew Hu, Brian Keith, Celeste Simon. Investigating endoplasmic reticulum stress responses in pancreatic ductal adenocarcinoma under lipid imbalance [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B010.

Abstract A058: Meta-analysis of single-cell RNA expression in genetically engineered mouse models of pancreatic ductal adenocarcinoma reveals inter-model heterogeneity

Abstract Background: Genetically engineered mouse models (GEMMs) are widely used in the study of pancreatic ductal adenocarcinoma (PDAC) because of their immune-competent tumor microenvironment (TME); however, the extent to which particular GEMMs recapitulate the tumor and TME observed in the patient population has not been systematically evaluated. In this study, we integrate single-cell RNA sequencing (sc-RNA-seq) data from multiple studies and multiple GEMM backgrounds to identify differences in the cellular compositions of popular PDAC GEMMs. Methods: A total of 49,191 cells were used from three studies, including normal mouse pancreas (N=2) and five different GEMM backgrounds (N=16). Data curation, integration, and analysis were based on the Seurat pipeline in R. SingelCellNet was used to train a random forest model on manually labeled human sc-RNA-seq data from 20 patients. To enable cross-species use, the classifier was trained using only genes with both human and mouse homologues. Cells classified as neoplastic were further clustered to quantify the number of classical and basal-like cells based on signature gene expression levels. The ratio of these subtypes in each GEMM and the relationship between the modified genes in each model were examined. Results: Ad-hoc clustering and a human-cell-trained single-cell classifier showed 79% agreement in an integrated data set of PDAC GEMMs. Cells identified by both methods as tumor (8,303 cells, 17% of total) were assessed for PDAC tumor subtype via subsequent clustering analysis (basal-like or classical). When comparing the ratio of differently subtyped tumor cells, we identified stark differences between GEMM genetic backgrounds. Among five different models, KIC (KrasLSL−G12D/+Ink4a/Arffl/flPtf1aCre/+), KPPCN (KrasLSL−G12D/+Trp53fl/flPdx1Cre/+Nsdhlfl/fl), and pdx1-KPC (KrasLSL−G12D/+Trp53LSL-R172H/+Pdx1Cre/+) exhibited a higher proportion of basal-like PDAC cells compared to KPfC/KPPC (KrasLSL−G12D/+Trp53fl/flPdx1Cre/+) and ptf1a-KPC (KrasLSL−G12D/+Trp53LSL-R172H/+Ptf1aCre/+). Interestingly, in the KIC model, which was harvested at early and late time points (40 or 60 days), classical PDAC was overrepresented in early models, and basal-like PDAC was more prevalent in the older tumors. While sample sizes are limited in this study, in Pdx1 driven models, we observed a bias towards basal-like phenotype in GEMMs using the Trp53LSL-R172H/+ method compared to those with Trp53fl/fl. Conclusions: In a comparison of publicly available sc-RNA-seq, we highlight potential biases in the molecular subtypes that arise from specific PDAC GEMMs. Because of the known link between tumor subtype and therapeutic response, these results suggest translational work may benefit from GEMM selection that considers transcriptomic diversity. Citation Format: Yun Jae Yoo, Ki H Oh, Luke A. Torre-Healy, Richard A. Moffitt. Meta-analysis of single-cell RNA expression in genetically engineered mouse models of pancreatic ductal adenocarcinoma reveals inter-model heterogeneity [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A058.

Obesity Influences the Expression of Key Immunomodulators in Normal Human Breast Tissue, Basal-like Breast Cancer Patients, and Cell Lines.

Among the different components of the breast cancer microenvironment are adipocytes, which are mainly composed of differentiated adipocytes and adipose progenitors. The role of obesity in tumor progression has become a key topic in clinical studies, but the mechanics of this are still misunderstood. There is significant evidence of serum amyloid (SAA1), an acute-phase protein, being heavily expressed in inflamed, septic conditions. VTCN1 and VSIR, members of the immunoglobulin family, are key players in T-cell regulation. The present study investigates the differentially expressed genes caused by adipose-conditioned media on the novel triple-negative breast cancer cell lines MDA MB 231 and MDA MB 468. RNA sequencing of adipocyte-conditioned media (ACM)-treated MDA MB 231 and MDA MB 468 cells were analyzed and compared using the gene sequencing enrichment analysis database (GSEA). GSEA was also done on microarray data from obese, non-tumorous breast tissue patients (GSE:33526) to show significantly upregulated immunomodulators. Obesity was also shown to influence gene expression related to immune sensing and evasion in a dataset analysis of basal-like obese patients (GSE:79858). We showed obesity significantly upregulated immunomodulators related to immune suppression in non-tumorous, basal-like patients, as well as in novel basal-like TNBC cell lines.

Abstract PR010: Single-cell sequencing elucidates the effects of chemotherapy on cancer cell heterogeneity and the tumor microenvironment of human pancreatic adenocarcinoma

Abstract The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) is a complex ecosystem that drives tumor progression. Improving our understanding of the PDAC TME and its role in response to therapy via in-depth single cell characterization will have broad clinical implications for biomarker development and therapeutic design. In this study, we performed single-cell RNA sequencing on freshly collected human PDAC samples of primary (n=16) or metastatic (n=11) origin, either before (n=20) or after (n=7) chemotherapy. We found a heterogeneous mixture of basal and classical Moffitt cancer cell subtypes in all samples, along with distinct cancer-associated fibroblast (CAF) and tumor-associated macrophage (TAM) subpopulations. We identified the major CAF subpopulations as inflammatory CAFs (iCAFs) and myofibroblastic CAFs (myCAFs); within these subpopulations were a very few cells expressing immunogenic features which have previously been associated with antigen presenting CAFs (apCAFs). Tumor-associated macrophages (TAMs) could be categorized into two major subpopulations, C1QC+ TAMs or SPP1+ TAMs, each with distinct functional characteristics. For example, phagocytosis-associated gene sets were enriched in C1QC+ TAMs, while angiogenesis-associated gene sets were enriched in SPP1+ TAMs. Comparison of naïve and chemotherapy treated primary PDAC samples revealed that classical and basal-like cancer cells exhibited similar transcriptional responses to chemotherapy; this contrasts with some previous reports which posited a shift towards a basal-like transcriptional program among treated samples. We further noted that treated samples evinced fewer ligand-receptor interactions, particularly between TIGIT on CD8+ T cells and its ligand on cancer cells. We also identified TIGIT, not PD1, as the major inhibitory checkpoint molecule of CD8+ T cells in the PDAC TME. Altogether, our results suggest that chemotherapy impacts the PDAC TME and may further reduce response to immunotherapy. Citation Format: Daniel Weissinger, Emily A. Kawaler, Gregor Werba, Ende Zhao, Despoina Kalfakakou, Surajit Dhara, Grace Oh, Xiaohong Jing, Nina Beri, Lauren Khanna, Tamas Gonda, Paul E. Oberstein, Cristina Hajdu, Cynthia Loomis, Adriana Heguy, Mara H. Sherman, Amanda W. Lund, Theodore H. Welling, Igor Dolgalev, Aristotelis Tsirigos, Diane M. Simeone. Single-cell sequencing elucidates the effects of chemotherapy on cancer cell heterogeneity and the tumor microenvironment of human pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR010.

Abstract A003: Developmental parallels in pancreatic cancer subtypes revealed by single cell transcriptomics

Abstract Recent research into the transcriptional subtypes in pancreatic ductal adenocarcinoma (PDA) has demonstrated their importance in predicting survival and therapeutic response. The Basal-like subtype is enriched in metastases and generally predicts poorer prognosis, while the Classical subtype is defined by gene expression reflective of pancreatic progenitors. The two-subtype scheme can be further subdivided into five subtypes that help better reflect the vast transcriptional heterogeneity found in PDA tumors. Basal-like A tumors fare worse than Basal-like B tumors. KRAS wildtype tumors are enriched in Classical B, which have the best prognosis, while Classical A is more reflective of the two-subtype Classical program. Hybrid tumors, generally a mix of Basal-like B and Classical A, are intermediate in prognosis between the Classical and Basal-like subtypes. Despite the importance of the subtypes, we have yet to fully understand how subtype programs within single cells of a tumor are intrinsically regulated, and how they contribute to tumor-level transcriptional observations. To investigate, we generated and leveraged a single cell RNA sequencing dataset enriched for malignant tumor cells spanning both primary and metastatic disease. Along with the richly annotated COMPASS dataset featuring bulk RNA sequencing of over 300 laser captured samples, we developed a single sample classifier utilizing gene pairs for assigning Basal-like A, Basal-like B, Hybrid, Classical A or Classical B to bulk tumors as well as individual cells, allowing interrogation of the subtypes across modalities. We find that the bulk subtype of the tumor is generally reflected in the dominant cell subtype. However, there are frequently cells of other subtypes within individual tumors, and substantial differences in cell subtype composition across tumors of the same subtype. In addition, we find that the proportion of Basal-like cells is a significant risk prognosticator. Investigating deeper into the transcriptional drivers of the subtypes reveals differences in expression of programs linked to endodermal development. Transcription factors such as FOXC1 and IRX3 generally related to foregut development are enriched in Basal-like A, forming a gradient of high expression to lower expression from Basal-like A, Basal-like B, Hybrid, Classical A to Classical B. Interestingly, midgut transcription factors such as CDX2 and ELF3 are expressed in the reverse order. Integrating data from single cell endoderm RNA sequencing further supports this finding, suggesting that the subtypes may arise from tumor cells exploiting pathways normally expressed early in development. This work provides a stepping stone to better understanding the mechanisms behind transcriptional subtypes in PDA. Citation Format: Sabrina Ge, Gun Ho Jang, Michelle Chan-Seng-Yue, Eugenia Flores Figueroa, Karen Ng, Stephanie Ramotar, Anna Dodd, Grainne M. O'Kane, Julie M. Wilson, Jennifer J. Knox, Sandra E. Fischer, Steven Gallinger, Faiyaz Notta. Developmental parallels in pancreatic cancer subtypes revealed by single cell transcriptomics [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A003.

Abstract A010: Persister cell phenotypes contribute to poor patient outcomes after neoadjuvant chemotherapy in PDAC

Abstract The impact of neoadjuvant chemotherapy on tumor cells is largely unknown with resistance to therapy remaining a significant challenge. We analyzed 171 chemo-naïve and post-chemotherapy resected patient samples (chemoradiotherapy excluded) using RNAseq and multiplexed immunofluorescence. Neoadjuvant chemotherapy enriched for distinct neoplastic persister phenotypes expressing Classical and Basal-like biomarkers GATA6 and KRT17. The enrichment of GATA6HiClassical-like and KRT17Hi Basal-like cells in post-chemotherapy patient samples was associated with poor survival after mFOLFIRINOX, but not gemcitabine. CYP3A subfamily enzymes (CYP3A4 and CYP3A5) were expressed in GATA6Hi and KRT17Hi persister cells and can degrade mFOLFIRINOX constituent irinotecan. Taken together these data support a model in which pre-existing subpopulations of GATA6Hi (Classical) and/or KRT17Hi (Basal) neoplastic cells emerge after mFOLFIRINOX treatment by expressing CYP3A. The identification of CYP3A-enabled persister cell phenotypes is an important step in the design of effective therapies to overcome drug-tolerance and prevent recurrence after neoadjuvant therapy. Citation Format: Xu Zhou, Roma Kurilov, John P. Neoptolemos, Benedikt Brors, Kai Hu, Teresa Peccerella, Jing-Yu An, Stephanie Roessler, Katrin Pfütze, Angela Schulz, Stephan Wolf, Nicolas Hohmann, Ulrike Heger, Oliver Strobel, Simon T Barry, Christoph Springfeld, Christine Tjaden, Frank Bergmann, Markus Büchler, Thilo Hackert, Franco Fortunato, Peter Bailey. Persister cell phenotypes contribute to poor patient outcomes after neoadjuvant chemotherapy in PDAC [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A010.

Tumor suppressor DEAR1 regulates mammary epithelial cell fate and predicts early onset and metastasis in triple negative breast cancer

Triple negative breast cancer (TNBC) is a disease of poor prognosis, with the majority classified as the basal-like subtype associated with epithelial-mesenchymal transition and metastasis. Because basal breast cancers originate from proliferative luminal progenitor-like cells upon dysregulation of proper luminal differentiation, genes regulating luminal-basal transition are critical to elucidate novel therapeutic targets to improve TNBC outcomes. Herein we demonstrate that the tumor suppressor DEAR1/TRIM62 is a critical regulator of luminal cell fate. DEAR1 loss in human mammary epithelial cells results in significantly enhanced mammosphere formation that is accelerated in the presence of TGF-β/SMAD3 signaling. Mammospheres formed following DEAR1 loss are enriched for ALDH1A1 and CK5 expression, EpCAM−/CD49f+ and CD44high/24low basal-like epithelial cells, indicating that DEAR1 regulates stem/progenitor cell properties and luminal-basal progenitor transition. We show that DEAR1 maintains luminal differentiation as a novel ubiquitin ligase for SNAI2/SLUG, a master regulator driving stemness and generation of basal-like progenitor populations. We also identify a significant inverse correlation between DEAR1 and SNAI2 expression in a 103 TNBC case cohort and show that low DEAR1 expression significantly correlates with young age of onset and shorter time to metastasis, suggesting DEAR1 could serve as a biomarker to stratify early onset TNBCs for targeted stem cell therapies.

Basal Cell-derived WNT7A Promotes Fibrogenesis at the Fibrotic Niche in Idiopathic Pulmonary Fibrosis.

Loss of epithelial integrity, bronchiolarization, and fibroblast activation are key characteristics of idiopathic pulmonary fibrosis (IPF). Prolonged accumulation of basal-like cells in IPF may impact the fibrotic niche to promote fibrogenesis. To investigate their role in IPF, basal cells were isolated from IPF explant and healthy donor lung tissues. Single-cell RNA sequencing was used to assess differentially expressed genes in basal cells. Basal cell and niche interaction was demonstrated with the sLP-mCherry niche labeling system. Luminex assays were used to assess cytokines secreted by basal cells. The role of basal cells in fibroblast activation was studied. Three-dimensional organoid culture assays were used to interrogate basal cell effects on AEC2 (type 2 alveolar epithelial cell) renewal capacity. Perturbation was used to investigate WNT7A function invitro and in a repetitive bleomycin model invivo. We found that WNT7A is highly and specifically expressed in basal-like cells. Proteins secreted by basal cells can be captured by neighboring fibroblasts and AEC2s. Basal cells or basal cell-conditioned media activate fibroblasts through WNT7A. Basal cell-derived WNT7A inhibits AEC2 progenitor cell renewal in three-dimensional organoid cultures. Neutralizing antibodies against WNT7A or a small molecule inhibitor of Frizzled signaling abolished basal cell-induced fibroblast activation and attenuated lung fibrosis in mice. In summary, basal cells and basal cell-derived WNT7A are key components of the fibrotic niche, providing a unique non-stem cell function of basal cells in IPF progression and a novel targeting strategy for IPF.

OR30-3 Direct and VGLL1-Mediated Gene Regulation by GRHL2 During Mesenchymal to Epithelial Transition in Breast Cancer

Abstract The transcription factor (TF) GRHL2 controls normal epithelial differentiation in different tissues. It is expressed in all breast cancer subtypes except those with a mesenchymal phenotype (claudin-low subtype), where its ectopic expression leads to induction of a mesenchymal-epithelial transition (MET) (1). Here we explored the mechanisms by which GRHL2 induces MET in claudin-low breast cancer cell lines using a combination of ChIP-seq and RNAseq analyses upon modulation of GRHL2 expression in mesenchymal and epithelial cell lines. Direct DNA binding by GRHL2 was enriched in proximity of genes positively regulated in mesenchymal cell lines, which include several epithelial TFs as well as an array of epithelial genes. Biotinylation and mass spectrometry analysis of membrane proteins confirmed surfaceome remodeling via induction of membrane proteins with roles in cell-cell contacts and adhesion. Repression of mesenchymal genes was mediated in part by direct induction of miRNAs targeting mesenchymal transcription factors. In addition, GRHL2 strongly induced expression of the cofactor VGLL1, whose expression is high in the triple-negative (TNBC) basal-like subtype. VGLL1 overexpression in mesenchymal cell lines and suppression in basal-like TNBC cells confirmed its role in mediating part of GRHL2 signaling, in particular via interaction with and modulation of TEAD transcription factors. In addition, GRHL2 also interacted with VGLL1 upon its induction in mesenchymal cells, recruiting it to some of its binding motifs, suggesting a multilayered cross-talk between GRHL2 and TEAD factors. Together, our results indicate that GRHL2 acts as a master regulator in the induction of TNBC epithelial differentiation via direct upregulation of epithelial transcription factors, miRNAs and TEAD cofactor VGLL1, resulting in both upregulation of epithelial genes and suppression of mesenchymal genes.(1) Cieply B. et al., Cancer Res. 2013 Oct. 1;73 (20): 6299-309 Presentation: Tuesday, June 14, 2022 10:15 a.m. - 10:30 a.m.

High Expression of CD58 and ALDH1A3 Predicts a Poor Prognosis in Basal-like Breast Cancer.

CD58 is an immune adhesion molecule on the cellular surface. It was previously found that a high expression of CD58 predicted a poor prognosis of patients with lower-grade gliomas. Therefore, the aim of this paper was to investigate the association between CD58 and breast cancer. CD58 gene expression data downloaded from cBioPortal was compared between the different subtypes of breast cancer. Clinical prognosis was examined using Kaplan-Meier analysis and multivariable Cox regression analysis. The association between CD58 expression and immune cell infiltration was estimated using the TIMER 2.0 web platform. Finally, the tumour sphere formation of aldehyde dehydrogenase 1 (ALDH1)high basal-like breast cancer stem cells in which CD58 was knocked down using siRNA was measured. CD58 mRNA was mainly enriched in claudin-low and basal-like subtypes. The high expression of CD58 predicted a good prognosis in patients with luminal A and luminal B breast cancer. This prediction may be due to the association of immune cell infiltration with CD58. Notably, patients with luminal A breast cancer with a high expression of CD58 in association with ALDH1A3 exhibited a good prognosis; however, this did not apply to patients with basal-like breast cancer. The in vitro experiments revealed that knockdown of CD58 inhibited the tumour sphere formation ability of ALDH1high basal-like cancer cells. CD58 may function as a potential prognostic biomarker and therapeutic target in ALDH-positive basal-like cancer stem cells.

1722P The subtype-specific cell fitness of head and neck cancer is regulated by the YBX1 integration of oncogenic PI3K/mTOR signalling

Molecular subtyping of heterogeneous cancers has gained substantial biological interest although with limited translation to the clinic. In heterogeneous head and neck cancer (HNC), subtype-specific treatment regimens are currently missing. A multi-omic approach integrating whole-genome and single-cell transcriptome sequencing with reverse-phase protein array was employed in patient primary and metastatic tumours and human cell lines to investigate the subtype-specific molecular mechanisms as the therapeutic vulnerabilities in HNC. An integrated analysis of HNC subtypes using single-cell sequencing and proteome profiles revealed a hallmark epithelial-mesenchymal transition (EMT) signature within the epithelial cancer-cell population. The EMT signature was evident in the invasive mesenchymal subtype and surprisingly coincided with PI3K/mTOR pathway inactivation. Conversely, the EMT signature was suppressed in epithelial cancer cells of the basal subtype which exhibited hyperactive PI3K/mTOR oncogenic signalling. We further identified that phosphorylation of Y Box binding protein 1 (YBX1), downstream of the PI3K/mTOR pathway, restrained the basal-like cancer cell proliferation. In contrast, YBX1 acted as a safeguard against the proliferation to invasion switch in mesenchymal-like epithelial cancer cells, and its loss accentuated partial EMT acquisition and in vivo invasion. Interestingly, the proteomic analysis of patient HNC identified phospho-YBX1 as a prognostic marker for overall patient outcomes, the loss of which correlated with HNC metastasis and poor prognosis. Our results indicate that HNC cells adopt a PI3K-phospho-YBX1-dependent proliferation program that is mutually exclusive to partial EMT involvement which relies on the lack of phosphorylation of YBX1 in PI3K-inactive cancer cells. These findings create a unique opportunity to sensitise mesenchymal cancer cells to PI3K/mTOR inhibitors by shifting them towards a basal-like subtype, constituting a promising therapeutic approach against HNC.

Decellularization of xenografted tumors provides cell-specific in vitro 3D environment.

In vitro cell culture studies are common in the cancer research field, and reliable biomimetic 3D models are needed to ensure physiological relevance. In this manuscript, we hypothesized that decellularized xenograft tumors can serve as an optimal 3D substrate to generate a top-down approach for in vitro tumor modeling. Multiple tumor cell lines were xenografted and the formed solid tumors were recovered for their decellularization by several techniques and further characterization by histology and proteomics techniques. Selected decellularized tumor xenograft samples were seeded with the HCC1806 human triple-negative breast cancer (TNBC) basal-like subtype cell line, and cell behavior was compared among them and with other control 2D and 3D cell culture methods. A soft treatment using Freeze-EDTA-DNAse allows proper decellularization of xenografted tumor samples. Interestingly, proteomic data show that samples decellularized from TNBC basal-like subtype xenograft models had different extracellular matrix (ECM) compositions compared to the rest of the xenograft tumors tested. The in vitro recellularization of decellularized ECM (dECM) yields tumor-type-specific cell behavior in the TNBC context. Data show that dECM derived from xenograft tumors is a feasible substrate for reseeding purposes, thereby promoting tumor-type-specific cell behavior. These data serve as a proof-of-concept for further potential generation of patient-specific in vitro research models.

Loss of FGFR4 promotes the malignant phenotype of PDAC

Transcriptomic analyses of pancreatic ductal adenocarcinoma (PDAC) have identified two major epithelial subtypes with distinct biology and clinical behaviours. Here, we aimed to clarify the role of FGFR1 and FGFR4 in the definition of aggressive PDAC phenotypes. We found that the expression of FGFR4 is exclusively detected in epithelial cells, significantly elevated in the classical PDAC subtype, and associates with better outcomes. In highly aggressive basal-like/squamous PDAC, reduced FGFR4 expression aligns with hypermethylation of the gene and lower levels of histone marks associated with active transcription in its regulatory regions. Conversely, FGFR1 has more promiscuous expression in both normal and malignant pancreatic tissues and is strongly associated with the EMT phenotype but not with the basal-like cell lineage. Regardless of the genetic background, the increased proliferation of FGFR4-depleted PDAC cells correlates with hyperactivation of the mTORC1 pathway both in vitro and in vivo. Downregulation of FGFR4 in classical cell lines invariably leads to the enrichment of basal-like/squamous gene programs and is associated with either partial or full switch of phenotype. In sum, we show that endogenous levels of FGFR4 limit the malignant phenotype of PDAC cells. Finally, we propose FGFR4 as a valuable marker for the stratification of PDAC patients.

Clinicopathological features of mixed cervical carcinoma with adenoid cystic pattern

Objective: To investigate the clinicopathological characteristics, immunophenotype, molecular characteristics, differential diagnosis, clinical treatment and prognosis of mixed carcinoma of cervix with adenoid cystic pattern. Methods: Three cases of mixed cervical carcinoma with adenoid cystic pattern were collected at the Affiliated Hospital of Xuzhou University Medical School from 2018 to 2021.The clinicopathological characteristics were analyzed, immunohistochemistry (IHC) and in situ hybridization (ISH) were performed. The related literature was reviewed. Results: The three patients were postmenopausal women with a median age of 74.7 years. The clinical symptom was vaginal bleeding without obvious causes. One case was an endophytic tumor, and the others were exophytic. The median diameter of the three cases was 3.3 cm. Two patients underwent hysterectomy, the tumors infiltrated the external 1/3 and middle 1/3 of the cervix respectively. All the lymph nodes were negative. One patient had a previous biopsy. Microscopically, all three tumors were characterized by a cribriform structure, which were filled with basophilic myxoid substance and surrounded by tubules lined by two layers of cells. The tumor cells had scanty cytoplasm and showed the characteristics of cervical basal-like cells. All three cases were accompanied by high-grade squamous intraepithelial lesions and squamous cell carcinoma, and one also showed a non-specific spindle cell sarcomatoid component. Within the double-layered epithelial structure, the outer epithelium was positive for p63, CD117, p16INK4a (clone E6H4) and MYB protein and negative for S-100 by IHC. The combined positive score of PD-L1 (clone 22C3) was less than 1 in all three cases. Human papillomavirus (HPV) types 16 and 18 were detected in one patient preoperatively, while high-risk HPV were positive in the other two patients by RNAscope ISH postoperatively. None of the three cases showed MYB gene rearrangement by FISH. The mean follow-up time was 23.3 months (36, 28 and 6 months, respectively). Two patients underwent hysterectomy and radiotherapy survived without disease. One patient survived with tumor just by radiotherapy and drug therapy. Conclusions: Mixed cervical carcinoma with adenoid cystic pattern is extremely rare. It is a high-grade malignancy with poor prognosis. The tumor is associated with high-risk HPV infection, without MYB gene rearrangement, and with low PD-L1 immunoreactivity. Radical surgery combined with radiotherapy and chemotherapy is the mainstay of treatment at present.

The Tumor-Specific Expression of L1 Retrotransposons Independently Correlates with Time to Relapse in Hormone-Negative Breast Cancer Patients.

Background: Long-Interspersed Nuclear Element (L1) retrotransposons are silenced in healthy tissues but unrepressed in cancer. Even if L1 reactivation has been associated with reduced overall survival in breast cancer (BC) patients, a comprehensive correlation with clinicopathological features is still missing. Methods: Using quantitative, reverse-transcription PCR, we assessed L1 mRNA expression in 12 BC cells, 210 BC patients and in 47 normal mammary tissues. L1 expression was then correlated with molecular and clinicopathological data. Results: We identified a tumor-exclusive expression of L1s, absent in normal mammary cells and tissues. A positive correlation between L1 expression and tumor dedifferentiation, lymph-node involvement and increased immune infiltration was detected. Molecular subtyping highlighted an enrichment of L1s in basal-like cells and cancers. By exploring disease-free survival, we identified L1 overexpression as an independent biomarker for patients with a high risk of recurrence in hormone-receptor-negative BCs. Conclusions: Overall, L1 reactivation identified BCs with aggressive features and patients with a worse clinical fate.

Abstract 1602: Lineage plasticity enables low ER luminal tumors to evolve and gain basal-like traits

Abstract Stratifying breast cancer into specific molecular or histological subtypes aids in therapeutic decision-making and predicting outcomes, however, these subtypes may not be as distinct as previously thought. Patients with luminal-like, Estrogen Receptor (ER)-expressing tumors have better prognosis than patients with more aggressive, triple-negative or basal-like tumors. There is, however, a subset of luminal-like tumors that express lower levels of ER, which exhibit more basal-like features. Previous studies have suggested that triple negative, basal-like tumors may arise from a luminal cell-of-origin, but there are no definitive studies that identify the cell-of-origin of these low ER tumors. Analysis of 2208 invasive breast carcinomas from 2012-2020 revealed that 2% of tumors have low ER expression (less than 10% ER positive cells), which are mostly high-grade carcinomas and exhibit basal-like features. TCGA analysis revealed that tumors with lower ER expression (lowest quartile of ER expression) expressed higher basal signature genes as compared to tumors with higher levels of ER expression. This variation within the ER+ subtype and the emergence of basal-like characteristics within low ER tumors suggest that some luminal tumors may evolve into a more basal-like or triple-negative subtype. The luminal mouse mammary tumor, MMTV-PyMT, was used to model low ER human tumors and, similar to the patient tumor samples, basal-like tumor cells were also found within these tumors. Lineage tracing using tissue-specific and inducible Cre recombinase-based labelling was performed to elucidate the lineage-of-origin of these basal-like cells, revealing that these basal-like cells were derived from normal luminal epithelial cells, not basal cells. Our study uncovers the existence of luminal-basal plasticity within tumors of a low ER subtype that enables these cells to transition into a more basal-like state. Understanding the factors that enable this plasticity to occur may reveal opportunities to curb the evolution of more aggressive traits, potentially improving the way breast cancer is currently managed and treated. Citation Format: Gadisti Aisha Nurulhijjah Binti Mohamed, Nevena B. Ognjenovic, Sundis Mahmood, Sarah Min Kyung Lee, Brock C. Christensen, Kristen E. Muller, Diwakar R. Pattabiraman. Lineage plasticity enables low ER luminal tumors to evolve and gain basal-like traits [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 1602.

Abstract 3644: Schwann cells reshape tumor cells and cancer associated fibroblasts in pancreatic ductal adenocarcinoma microenvironment

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by high degree of innervation and desmoplasia in its tumor microenvironment (TME). Nerves and cancer associated fibroblast (CAFs) represent two key components of TME. Schwann cells (SCs), the most prevalent cell type in peripheral nerves, have been reported to support neurons and facilitate neuronal guidance during repair, but their role and mechanism in PDAC development remain largely unknown. Cancer-associated fibroblasts (CAFs) are believed to be mainly responsible for desmoplasia in PDAC TME, but whether they are also involved in the regulation of SCs functions in PDAC is yet to be explored. Here, we aimed to illustrate the function and underlying mechanism of SCs in PDAC TME and verified its clinical relevance. Methods: We performed tissue microarray to evaluate the enrichment of SCs in tumors of different stages, as well as in adjacent normal tissues. Single cell RNA-sequencing (scRNA-seq) and microarray-based spatial transcriptomics (ST) were used to determine the interactions among SCs, tumor cells, and CAFs. We utilized co-culture and conditioned medium (CM) model to evaluate the role of SCs in reshaping TME. We employed intrasplenic injection model and patient-derived xenograft (PDX) mouse models to determine the role of SCs in tumor progression. To evaluate the differentiation of CAFs after SCs CM treatment, immunohistochemistry (IHC), immunofluorescence (IF), Western blot, enzyme-linked immunosorbent assay (ELISA), qPCR and bulk-RNA seq were performed. Mass spectrometry (MS), ELISA, and IF assays were also performed to identify the key regulators of SCs. Results: Significant neural hypertrophy and SCs infiltration were observed in tumor tissues and were associated with worse prognosis in PDAC patients. More cancer cells and CAFs could also be seen in proximity to SCs. scRNA-seq of PDAC samples demonstrated high degree of heterogeneity in both cancer cells and CAFs. We found a basal-like subgroup of cancer cells with more malignant behavior, and a pro-inflammatory subpopulation of CAFs (iCAFs) that promotes tumor progression. ST further showed that both basal-like cancer cells and iCAFs were located immediately adjacent to SCs in human PDAC tissues. The malignant phenotypes of cancer cells including migration, invasion, and proliferation were significantly promoted by SCs CM treatment. Furthermore, SCs CM treatment promotes iCAFs formation and PDAC progression in vitro and in vivo. Lastly, IL-1α was identified as a SCs-secreted ligand that generate iCAFs. Conclusion: Collectively, we find that SCs play an oncogenic role in PDAC by promoting the shift of cancer cells and CAFs toward more malignant phenotypes, and suggest that targeting SCs in TME represents a promising therapeutic strategy for PDAC treatment. Citation Format: Meilin Xue, Yongsheng Jiang, Lijie Han, Rui Su, Cheng Xiong, Youwei Zhu, Yizhi Cao, Minmin Shi, Jianjun Chen, Baiyong Shen, Lingxi Jiang, Hao Chen. Schwann cells reshape tumor cells and cancer associated fibroblasts in pancreatic ductal adenocarcinoma microenvironment [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 3644.

Abstract LB127: Reduced expression of annexin-a6 induces metabolic reprogramming that favors rapid fatty acid oxidation in triple-negative breast cancer cells

Abstract The ability of cancer cells to alter their metabolism is one of the major mechanisms underlying rapid tumor progression and/or therapeutic resistance in solid tumors, including the hard-to-treat triple-negative breast cancer (TNBC) subtype. Here, we assessed the contribution of the tumor suppressor, Annexin A6 (AnxA6) in the metabolic adaptation of rapidly growing versus invasive TNBC cells as well as in Lapatinib resistant (Lap-R) TNBC cells. AnxA6 expression in cultured TNBC cell lines, Lap-R cells and in crude isolates of mitochondria was determined by Western blotting and/or immunofluorescence assays. The oxygen consumption rates (OCR), extracellular acidification rates (ECAR) and mitochondria activity were assessed using the Seahorse XF Analyzer. Intracellular metabolites were detected by 1H-NMR analysis. Using model proliferative basal-like and invasive mesenchymal-like TNBC cell lines, we show that TNBC cells also exhibit metabolic heterogeneity. Down regulation of AnxA6 in TNBC cells attenuated mitochondrial respiration, glycolytic flux, and cellular ATP production capacity, resulting in a quiescent cellular energy phenotype. We further show that AnxA6-depletion was associated with decreased lipid droplet accumulation and the lipolytic phenotype by enhancing the uptake and mitochondrial fatty acid oxidation for ATP production. Chronic Lapatinib-induced up regulation of AnxA6 in AnxA6-low TNBC cells reversed their lipolytic to a more lipogenic/glycolytic metabolic phenotype with gluconeogenic precursors as additional metabolites. Collectively, these data suggest that the expression status of AnxA6 in TNBC cells underlies significant metabolic adaptation during stress and/or chronic Lapatinib treatment and provide additional insights into the potential of AnxA6 as a biomarker for not only therapeutic intervention but also the metabolic subtyping of TNBC subsets. Citation Format: Stephen D. Williams, Sarrah E. Widatalla, Amos M. Sakwe. Reduced expression of annexin-a6 induces metabolic reprogramming that favors rapid fatty acid oxidation in triple-negative breast cancer cells [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 LB127.

MP06-08 SINGLE-CELL SEQUENCING OF PARENTAL BLADDER CANCER AND SUBSEQUENT ORGANOIDS REVEALS DIFFERENTIAL CELL PRESERVATION

You have accessJournal of UrologyCME1 May 2022MP06-08 SINGLE-CELL SEQUENCING OF PARENTAL BLADDER CANCER AND SUBSEQUENT ORGANOIDS REVEALS DIFFERENTIAL CELL PRESERVATION Roland Seiler, Martina Minoli, Franziaks Singer, Anne Bertolini, Ulrike Menzel, Christian Beisel, and Marianna Kruithof-de Julio Roland SeilerRoland Seiler More articles by this author , Martina MinoliMartina Minoli More articles by this author , Franziaks SingerFranziaks Singer More articles by this author , Anne BertoliniAnne Bertolini More articles by this author , Ulrike MenzelUlrike Menzel More articles by this author , Christian BeiselChristian Beisel More articles by this author , and Marianna Kruithof-de JulioMarianna Kruithof-de Julio More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002523.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Molecular heterogeneity of bladder cancer (BC) is affecting response to treatment. To test treatment response, several patient derived models have been established that may vary in their suitability to test targets. We have previously established patient derived organoids (PDO) from BC patients and found a highly conserved genomic landscape. In this analysis, we aim to investigate the cellular composition and pathway activities of this patient derived model. METHODS: We generated PDOs from more than 35 BC specimens. Based on the marker expression, we selected 5 pairs of BC and subsequent PDO to include different molecular subtypes (e.g. luminal-, basal-like). Single-cell suspensions were directed to scRNA-seq technology from 10x Genomics. For the analysis, unsupervised clustering, gene expression, as well as pathway analysis were used to compare the cell type composition and pathway activity. RESULTS: Overall, we observed a high degree of heterogeneity of cell type composition between BCs, between PDOs, as well as between BCs to subsequent PDOs. Unsupervised clustering separated cancerous- as well as non-cancerous cells in BCs and PDOs in individual clusters. In most BC, both luminal-like and basal-like cells were detected. In the subsequent PDOs however, luminal-like cells were present with a higher proportion compared to basal-like cells. This was true for different luminal-like subcategories (lum-pap and lum-nonspecified). While immune cells (e.g. B-cells, CD4+ and CD8+ T-cells) were present in both BC and PDO samples, stromal cells (e.g. smooth muscle and endothelial cells) were absent in PDOs. Consequently, immune- and cancer cell related pathways such as interferon_gamma_response or FGFR3_signature were active in both BC and PDO. Contrarily, pathways related to stroma interactions such as angiogenesis or epithelial_mesenchymal_transition were less active in PDOs. CONCLUSIONS: Single-cell transcriptomic profiling of parental BCs and subsequent PDOs revealed a high degree of heterogeneity in both the tumor and organoid cellular composition. Our data show preservation of immune cells in PDOs and thus suggest that organoids may even allow to investigate cellular interactions between cancer and immune cells. On the other hand, since stromal cells were mainly lost in PDOs, other models may be needed to investigate angiogenesis or epithelial mesenchymal transition. Source of Funding: SNSF Grant Nr. 310030 Novartis Grant #19C200grant #2017-510 of the Strategic Focal Area “Personalized Health and Related Technologies (PHRT)” of the ETH Domain © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e79 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Roland Seiler More articles by this author Martina Minoli More articles by this author Franziaks Singer More articles by this author Anne Bertolini More articles by this author Ulrike Menzel More articles by this author Christian Beisel More articles by this author Marianna Kruithof-de Julio More articles by this author Expand All Advertisement PDF DownloadLoading ...

Epitheliosis is a histopathological finding associated with malignancy and poor prognosis in dogs with mammary tumors.

Canine mammary epitheliosis (ME) is a poorly studied dysplasia that may have premalignant potential. In this study, the clinicopathological relevance of ME was prospectively studied in 90 female dogs with mammary tumors (MTs) that underwent radical mastectomy. ME distribution, extent, and coexistence with benign and malignant MTs were evaluated for each case (505 mammary glands). ME was macroscopically undetectable and was present in 47/90 (52%) cases, frequently bilateral. In dogs with malignant MTs and ME, diffuse ME throughout the mammary chain was present in 10/39 (26%) cases. A histological ME-carcinoma transition was evident in certain histotypes. By immunohistochemistry (AE1/AE3, cytokeratin 14 [CK-14], CK-8/18, vimentin, calponin, p63, Ki-67, estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2), ME was a slow-growing, triple-negative process with a strong predominance of basal-like nonmyoepithelial cells. ME was associated with older dogs (P = .016), malignant tumors (P = .044), worse clinical stages (P = .013), lymph node metastasis (LNM, P = .021), higher histological grade tumors (P = .035), and shorter overall survival (OS) in univariate analysis (P = .012). Interestingly, ME was distantly located to the malignant tumor in most cases (P = .007). In multivariate analyses, LNM (P = .005), histological grade (P = .006), and tumor size (P = .006) were independent predictors of OS. For the pathologist, the observation of ME should be clearly stated in the MT biopsy report to alert the surgeon/oncologist. Given the differences between canine ME and its human histopathological counterpart (atypical ductal hyperplasia), "epitheliosis" should remain the preferred term for the dog.