Metastatic Subpopulations Research Articles

8061 Factors Affecting Survival of Metastatic Medullary Thyroid Cancer: A Population-Based Study From Surveillance, Epidemiology, and End Results (SEER) Program

Abstract Disclosure: A. Abdelhameed: None. H. Ibrahim: None. M. Mortagy: None. Introduction: Medullary thyroid cancer (MTC) arises from parafollicular cells of the thyroid. 43% of patients with MTC have metastasis. It was shown that the overall 5-year survival of patients with MTC is 40.4%. The clinical course of MTC is highly variable. The Aim is to identify factors affecting survival of the metastatic MTC sub-population. Understanding these factors is important in treating these patients. Methods 340 patients: with metastatic MTC who were diagnosed from 1975 to 2020 were extracted from SEER after removing duplicates and those not diagnosed by positive histology or cytology. Survival analysis was conducted, and Kaplan-Meier graph was generated. Univariate Cox regression was performed for all variables and respective hazard ratios (HR) were produced. Multivariable cox regression was performed for statistically significant variables. Results: The cohort were 59% males with a median age of 60 years. Median survival for the cohort was 20 months with survival of 63%, 41% and 31% at 1, 3, and 5 years respectively. Univariate Cox regression was statistically significant for age (HR 1.02), brain metastasis (HR 2.70), liver metastasis (1.65), lung metastasis (1.42), not performing total thyroidectomy (HR 2.77), lymph node ratio (HR 2.45), and systemic therapy after surgery (HR 0.34). Univariate Cox regression was not statistically significant for sex, grade, radiotherapy, chemotherapy, treatment delay, bone metastasis, distant lymph node metastasis, multifocal cancer, total number of benign and malignant tumors, race/ethnicity, income, tumor size and living in rural areas. Multivariable cox regression was done after inclusion of all significant variables from univariate Cox regression. It was only significant for age (HR 1.04), presence of lung metastasis (HR 1.91), and systemic therapy before surgery (HR 14.41). Conclusion: Increasing age, presence of lung metastasis and systemic therapy before surgery lead to worse prognosis in metastatic MTC. Gender, grade, radiotherapy, chemotherapy, treatment delay, bone or brain or liver metastasis, presence of multifocal cancer, race, tumor size and surgery don't affect survival according to multivariable cox regression. Future research is needed to study this group of patients. Presentation: 6/2/2024

Abstract 1550: Identification of a novel squalene epoxidase function in breast cancer brain metastasis

Abstract Brain metastases are serious complications of breast cancer, especially in triple-negative breast cancer (TNBC) patients. There is currently no effective treatment due to the unique brain microenvironment and limitation for drugs to cross the blood-brain barrier (BBB). BBB limits access of nutrients from the circulation and thereby makes the brain hypoxic and depleted of metabolites, growth factors and proteins. However, it remains poorly understood how breast cancer cells manipulate for adaption and colonization in the brain. To probe the molecular mechanism of breast cancer brain metastasis (BCBM), we compared gene signatures in primary breast tumors and BCBM tumors, and identified that SQLE (encodes squalene epoxidase, the second rate-limiting enzyme in the cholesterol biosynthesis) could be a leading-edge gene in breast cancer brain metastasis. To explore the role of SQLE in BCBM, we established the brain metastatic TNBC MDA-MB-231 (MDA231-BrM) cell line from brain metastatic subpopulation that originated from its parental MDA-MB-231 (MDA231) cell line. We found that SQLE expression was greatly upregulated in MDA231-BrM cells compared with the parental MDA231 cells. Using MDA231-BrM and MDA231 cell lines expressing SQLE shRNAs, we evaluated the effects of SQLE loss on cancer cell migration, invasion, and stemness by wound-healing, transwell invasion/migration, and tumorsphere formation assays. While loss of SQLE greatly attenuated cell invasiveness and stemness in both MDA231 and MDA231-BrM cells, loss of SQLE could only affect the cell migration activity on MDA231 cells but not MDA231-BrM cells. Our RNA-seq data further identified a subset of SQLE-affected genes that is uniquely enriched in MDA231-BrM cells and favors brain extravasation and colonization. To explore the potential function of SQLE in brain extravasation and colonization, we established in vitro BBB models and ex vivo mouse brain slice organotypic cultures. We showed that loss of SQLE inhibited the ability of MDA231-BrM cells to across the BBB-mimic astrocyte-endothelial structures as well as impaired the co-option with blood capillaries in the mouse brain slices. Although SQLE-deficient MDA231-BrM cells could still spread on the surface of the blood vessels, they seemed to undergo apoptosis. Since the ability to invade, migrate, and penetrate is critical for invasion of cancer cells, our results strongly imply the novel function of SQLE in breast cancer cell invasion, penetration, and even colonization in the brain through blood vessel co-option. In summary, our data reveal a novel role for SQLE in two critical requisites for breast-to-brain extravasation and colonization - the ability to penetrate through BBB and to co-opt brain vessels for metastatic expansion. Our findings indicate that targeting SQLE may represent a therapeutic opportunity for breast cancer brain metastases. Citation Format: Shu-Ping Wang, Jia-Yun Yeh, Yu-Ling (Pony) Lee, Kun-Yuan Lin, Chao-Di Chang, Chih-Chieh Yang, Shan-Yun Cheng, Ya-Wen Hung, Yu-Hsien Chang, Yao-Feng Li, Yung-Lung Yu. Identification of a novel squalene epoxidase function in breast cancer brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1550.

Abstract 1153: Using spatial transcriptomics to dissect cell to cell cooperation in lung adenocarcinoma

Abstract Lung cancer is the second most common cancer in the United States and the leading cause of cancer related deaths in this country. Unfortunately, efficacious treatments for lung cancer remain suboptimal. Lung cancer is characterized by several distinct subtypes of which lung adenocarcinoma is the most common, comprising about 40% of this malignancy. The hallmark gene mutations of lung adenocarcinoma include TP53, RAS, and STK11. LKB1 is a serine-threonine kinase (coded by the gene STK11) that largely functions as a tumor suppressor, and is mutated in 20-30% of non-small cell lung cancers (NSCLCs). The diversity between and within lung cancer subtypes, as well as within a patient (i.e. metastases vs primary tumor), makes treating this cancer very challenging. Lung adenocarcinoma, in particular, has collective invasion packs of cells adjacent to the primary tumor that correlate with metastatic disease in mouse models. We hypothesize that the transcriptomic profile of the collective invasion packs in lung adenocarcinoma patients varies significantly from the adjacent primary tumor and represents a targetable metastatic sub-population. This work will help to identify specific cell signaling pathways that have the translational potential to develop novel therapeutics for metastatic disease, ultimately improving patient outcomes through precision medicine. Utilizing patient lung adenocarcinoma samples with KRAS and KRAS+LKB1 mutations, we identified regions of interest including bulk tumor and surrounding invasion packs. Then, using GeoMx digital spatial profiling technology (by Nanostring) and next-gen sequencing, we generated transcriptomic profiles from bulk tumor and invasion packs. Preliminary results indicate region specific transcriptomic differences, highlighting the heterogeneity of these cell populations. More specifically, collective invasion packs exhibit upregulation of gene networks involving immune cell differentiation, function, oxidative phosphorylation, tumor invasiveness, and mitochondrial structure. To discern the metastatic potential, transcriptomic profiles and biological functions will be compared between invasion packs, tumor bulk vs invasion packs, and finally, inter-patient differences. Successful completion of this project will characterize transcriptomes of metastatic lung cancer and has the potential to ultimately identify biomarkers of aggressive disease. Lastly, this foundational study will pave the way for future studies on the transcriptomic landscape of metastatic pediatric cancers and cancer predisposition syndromes. Citation Format: Raehannah Jamshidi, Lyra Griffiths, Rich Johnston, Vaunita Parihar, Frank Schneider, Adam Marcus. Using spatial transcriptomics to dissect cell to cell cooperation in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1153.

Cells in the liver microenvironment regulate the process of liver metastasis.

The research of liver metastasis is a developing field. The ability of tumor cells to invade the liver depends on the complicated interactions between metastatic cells and local subpopulations in the liver (including Kupffer cells, hepatic stellate cells, liver sinusoidal endothelial cells, and immune-related cells). These interactions are mainly mediated by intercellular adhesion and the release of cytokines. Cell populations in the liver microenvironment can play a dual role in the progression of liver metastasis through different mechanisms. At the same time, we can see the participation of liver parenchymal cells and nonparenchymal cells in the process of liver metastasis of different tumors. Therefore, the purpose of this article is to summarize the relationship between cellular components of liver microenvironment and metastasis and emphasize the importance of different cells in the occurrence or potential regression of liver metastasis.

Alternative Polyadenylation Characterizes Epithelial and Fibroblast Phenotypic Heterogeneity in Pancreatic Ductal Adenocarcinoma

Human tumors are characterized by extensive intratumoral transcriptional variability within the cancer cell and stromal compartments. This variation drives phenotypic heterogeneity, producing cell states with differential pro- and anti-tumorigenic properties. While bulk RNA sequencing cannot achieve cell-type-specific transcriptional granularity, single-cell sequencing has permitted an unprecedented view of these cell states. Despite this knowledge, we lack an understanding of the mechanistic drivers of this transcriptional and phenotypic heterogeneity. 3′ untranslated region alternative polyadenylation (3′ UTR-APA) drives gene expression alterations through regulation of 3′ UTR length. These 3′ UTR alterations modulate mRNA stability, protein expression and protein localization, resulting in cellular phenotypes including differentiation, cell proliferation, and migration. Therefore, we sought to determine whether 3′ UTR-APA events could characterize phenotypic heterogeneity of tumor cell states. Here, we analyze the largest single-cell human pancreatic ductal adenocarcinoma (PDAC) dataset and resolve 3′ UTR-APA patterns across PDAC cell states. We find that increased proximal 3′ UTR-APA is associated with PDAC progression and characterizes a metastatic ductal epithelial subpopulation and an inflammatory fibroblast population. Furthermore, we find significant 3′ UTR shortening events in cell-state-specific marker genes associated with increased expression. Therefore, we propose that 3′ UTR-APA drives phenotypic heterogeneity in cancer.

Warburg effect enhanced by AKR1B10 promotes acquired resistance to pemetrexed in lung cancer-derived brain metastasis

BackgroundResistance to pemetrexed (PEM), a rare chemotherapeutic agent that can efficiently cross the blood-brain barrier, limits the therapeutic efficacy for patients with lung cancer brain metastasis (BM). Aldo-keto reductase family 1 B10 (AKR1B10) was recently found to be elevated in lung cancer BM. The link between AKR1B10 and BM-acquired PEM is unknown.MethodsPEM drug-sensitivity was assessed in the preclinical BM model of PC9 lung adenocarcinoma cells and the BM cells with or without AKR1B10 interference in vitro and in vivo. Metabolic reprogramming of BM attributed to AKR1B10 was identified by chromatography-mass spectrometry (GC-MS) metabolomics, and the mechanism of how AKR1B10 mediates PEM chemoresistance via a way of modified metabolism was revealed by RNA sequencing as well as further molecular biology experimental approaches.ResultsThe lung cancer brain metastatic subpopulation cells (PC9-BrM3) exhibited significant resistance to PEM and silencing AKR1B10 in PC9-BrM3 increased the PEM sensitivity in vitro and in vivo. Metabolic profiling revealed that AKR1B10 prominently facilitated the Warburg metabolism characterized by the overproduction of lactate. Glycolysis regulated by AKR1B10 is vital for the resistance to PEM. In mechanism, AKR1B10 promoted glycolysis by regulating the expression of lactate dehydrogenase (LDHA) and the increased lactate, acts as a precursor that stimulates histone lactylation (H4K12la), activated the transcription of CCNB1 and accelerated the DNA replication and cell cycle.ConclusionsOur finding demonstrates that AKR1B10/glycolysis/H4K12la/CCNB1 promotes acquired PEM chemoresistance in lung cancer BM, providing novel strategies to sensitize PEM response in the treatment of lung cancer patients suffering from BM.

Disintegrin-like Protein Strategy to Inhibit Aggressive Triple-Negative Breast Cancer.

Venoms are a rich source of bioactive compounds, and among them is leberagin-C (Leb-C), a disintegrin-like protein derived from the venom of Macrovipera lebetina transmediterrannea snakes. Leb-C has shown promising inhibitory effects on platelet aggregation. Previous studies have demonstrated that this SECD protein specifically targets α5β1, αvβ3, and αvβ6 integrins through a mimic mechanism of RGD disintegrins. In our current study, we focused on exploring the potential effects of Leb-C on metastatic breast cancer. Our findings revealed that Leb-C disrupted the adhesion, migration, and invasion capabilities of MDA-MB-231 breast cancer cells and its highly metastatic D3H2LN sub-population. Additionally, we observed significant suppression of adhesion, migration, and invasion of human umbilical vein endothelial cells (HUVECs). Furthermore, Leb-C demonstrated a strong inhibitory effect on fibroblast-growth-factor-2-induced proliferation of HUVEC. We conducted in vivo experiments using nude mice and found that treatment with 2 µM of Leb-C resulted in a remarkable 73% reduction in D3H2LN xenograft tumor size. Additionally, quantification of intratumor microvessels revealed a 50% reduction in tumor angiogenesis in xenograft after 21 days of twice-weekly treatment with 2 µM of Leb-C. Collectively, these findings suggest the potential utility of this disintegrin-like protein for inhibiting aggressive and resistant metastatic breast cancer.

Single-cell RNA sequencing reveals a pro-metastatic subpopulation and the driver transcription factor NFE2L1 in ovarian cancer cells.

Although cytoreductive surgery followed by adjuvant chemotherapy is effective as a standard treatment for early-stage ovarian cancer, the majority of ovarian cancer cases are diagnosed at the advanced stages with dissemination to the peritoneal cavity, leading to a poor prognosis. Therefore, it is crucial to understand the cellular and molecular mechanisms underlying metastasis and identify novel therapeutic targets. In this study, we aimed to elucidate the mechanisms underlying gene expression alterations during the acquisition of metastatic potential and characterize the metastatic subpopulations within ovarian cancer cells. We conducted single-cell RNA sequencing of two human ovarian cancer cell lines: SKOV-3 and SKOV-3-13, a highly metastatic subclone of SKOV-3. Suppression of NFE2L1 expression was performed through siRNA-mediated knockdown and CRISPR-Cas9-mediated knockout. Clustering and pseudotime trajectory analysis revealed pro-metastatic subpopulation within these cells. Furthermore, gene set enrichment analysis and prognosis analysis indicated that NFE2L1 could be a key transcription factor in the acquisition of metastasis potential. Inhibition of NFE2L1 significantly reduced migration and viability of both cells. In addition, NFE2L1 knockout cells exhibited significantly reduced tumor growth in a mouse xenograft model, recapitulating in silico and in vitro results. The results presented in this study deepen our understanding of the molecular pathogenesis of ovarian cancer metastasis with the ultimate goal of developing treatments targeting pro-metastatic subclones prior to metastasis.

Abstract B041: Bone microenvironment cues shift Ewing sarcoma cells towards pro-metastatic, ECM-secreting cell states

Abstract Solid tumors are complex ecosystems comprised of subpopulations of heterogeneous cancer cells that are altered by interactions with diverse cellular and acellular factors present in tumor microenvironments (TMEs). Accruing evidence suggests that heterogeneity of tumor cells and the TME, and bidirectional crosstalk between these spatially and temporally dynamic components, contributes to the generation of metastatic and therapy-resistant cell subpopulations. Ewing sarcomas are highly metastatic bone and soft tissue tumors driven by pathognomonic FET:ETS transcription factor fusions, predominantly EWS::FLI1. Despite sharing this single oncogenic driver, we and others have shown that Ewing sarcoma tumor cells are highly plastic cells that exist in transcriptionally and phenotypically distinct states. Notably, the extent of EWS::FLI1 transcriptional activity, ranging from low to high, has been shown to in part define the Ewing sarcoma cell state. Cells with high levels of fusion protein activity are generally proliferative and non-motile, while cells with lower levels of EWS::FLI1 expression or activity display enhanced metastatic potential. In this study we have leveraged single cell mRNA-seq and CITE-seq of Ewing sarcoma cell lines, gene expression and immunohistochemical profiling of primary tumor samples, and 3D culture invasion assays to define the complex Ewing sarcoma ecosystem and to investigate the role of tumor cell subpopulations and tumor: TME cross talk on tumor biology. Our results show cells with low levels of EWS::FLI1 activity activate an epithelial-mesenchymal transition (EMT)-like shift in gene expression. This activation of a partial EMT program is notable for an increase in expression and secretion of extracellular matrix (ECM) proteins by subpopulations of tumor cells and by increased matrix invasion. These ECM-related genes serve as transcriptional markers of the EMT-like cell state in both Ewing sarcoma models and in primary patient tumors. Moreover, the local TME surrounding these subpopulations is rich in tumor cell-derived ECM proteins including Tenascin-C, biglycan, and SPARC, proteins implicated in promotion of metastasis. We propose that subpopulations of ECM-secreting Ewing sarcoma tumor cells remodel the TME in a manner reminiscent of cancer associated fibroblasts (CAFs) in carcinomas. Further, we propose that this CAF-like matrix deposition contributes to local tumor progression and metastatic dissemination. Significantly, this ECM-secreting tumor cell state can be induced by canonical Wnt and TGF-beta ligands, suggesting that factors present in the bone TME can serve as inducers of a metastatic cell state. Together these findings demonstrate that Ewing sarcoma metastasis may be promoted by specific cell subpopulations which respond to and remodel the bone tumor microenvironment. Citation Format: Emma Wrenn, April Apfelbaum, Trisha Lipson, Erin Rudzinski, Xuemei Deng, Katherine Braun, Nicolas Garcia, Shruti Bhise, Olivia Waltner, Scott Furlan, Beth Lawlor. Bone microenvironment cues shift Ewing sarcoma cells towards pro-metastatic, ECM-secreting cell states [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B041.

Highly Metastatic Subpopulation of TNBC Cells Has Limited Iron Metabolism and Is a Target of Iron Chelators.

Excess iron is known to be a risk factor of carcinogenesis. Although iron chelators show anti-cancer effects, they have not been used successfully to treat cancer patients. Triple-negative breast cancer (TNBC) is a disease with poor prognosis without effective treatments. Thus, we aimed to evaluate a possibility of iron chelators as a therapy for TNBC. Deferasirox (DFX), an iron chelator, suppressed the growth of 4T1 murine TNBC cell line cells in vitro and in vivo. Lung metastasis was further significantly reduced, leading to the hypothesis that iron metabolism between metastatic and non-metastatic cells may be different. An analysis of existing database demonstrated that the expression of iron-uptake genes was significantly suppressed in TNBC cells that metastasized to lymph nodes or lungs compared to those in primary tumors. A highly metastatic clone of the murine 4T1 TNBC cells (4T1-HM) did not proliferate well under iron-rich or iron-depleted conditions by iron chelators compared to a low-metastatic clone (4T1-LM). Bulk RNA-seq analysis of RNA from 4T1-HM and 4T1-LM cells suggested that the PI3K-AKT pathway might be responsible for this difference. Indeed, DFX suppressed the proliferation via the AKT-mTOR pathway in 4T1-HM and the human MDA-MB-231 cells, a human mesenchymal-like TNBC cell line. DFX also suppressed the growth of 4T1-HM tumors in comparison to 4T1-LM tumors, and reduced lung metastases after surgical resection of primary 4T1 tumors. These results indicated, for the first time, that highly metastatic TNBC cells have limited iron metabolism, and they can be more effectively targeted by iron chelators.

LAMC2 marks a tumor-initiating cell population with an aggressive signature in pancreatic cancer

BackgroundTumor-initiating cells (TIC), also known as cancer stem cells, are considered a specific subpopulation of cells necessary for cancer initiation and metastasis; however, the mechanisms by which they acquire metastatic traits are not well understood.MethodsLAMC2 transcriptional levels were evaluated using publicly available transcriptome data sets, and LAMC2 immunohistochemistry was performed using a tissue microarray composed of PDAC and normal pancreas tissues. Silencing and tracing of LAMC2 was performed using lentiviral shRNA constructs and CRISPR/Cas9-mediated homologous recombination, respectively. The contribution of LAMC2 to PDAC tumorigenicity was explored in vitro by tumor cell invasion, migration, sphere-forming and organoids assays, and in vivo by tumor growth and metastatic assays. mRNA sequencing was performed to identify key cellular pathways upregulated in LAMC2 expressing cells. Metastatic spreading induced by LAMC2- expressing cells was blocked by pharmacological inhibition of transforming growth factor beta (TGF-β) signaling.ResultsWe report a LAMC2-expressing cell population, which is endowed with enhanced self-renewal capacity, and is sufficient for tumor initiation and differentiation, and drives metastasis. mRNA profiling of these cells indicates a prominent squamous signature, and differentially activated pathways critical for tumor growth and metastasis, including deregulation of the TGF-β signaling pathway. Treatment with Vactosertib, a new small molecule inhibitor of the TGF-β type I receptor (activin receptor-like kinase-5, ALK5), completely abrogated lung metastasis, primarily originating from LAMC2-expressing cells.ConclusionsWe have identified a highly metastatic subpopulation of TICs marked by LAMC2. Strategies aimed at targeting the LAMC2 population may be effective in reducing tumor aggressiveness in PDAC patients. Our results prompt further study of this TIC population in pancreatic cancer and exploration as a potential therapeutic target and/or biomarker.

Abstract P097: Comparative single cell transcriptome profiling of primary tumors, CTCs and metastatic sites from a bladder cancer PDX model

Abstract Background: A PDX bladder cancer model, BL0293-F563, grows large subcutaneous tumors, spontaneously metastasizes to the liver and bone, and sheds high numbers of circulating tumor cells (CTCs). This PDX model provides a unique opportunity to explore the relationships between primary tumors, CTCs and metastatic cell subpopulations. Methods: BL0293-F563 tumors (available from the NCI Patient-Derived Models Repository [https://pdmr.cancer.gov/] and originally developed by Jackson Laboratories) were implanted into NSG mice and and primary tumors, metastatic nodules in the liver, and blood were collected at maximal allowable tumor burden. Tumor tissue was dissociated using Miltenyi Tumor Dissociation Kit with OctoDissociator, and Human CTCs were enriched from whole mouse blood through negative selection with anti-mouse CD45 and anti-mouse MHC-1 magnetic beads. Single cell sequencing was done using 10X Genomics 3’ gene expression assay v3.1. Sequencing libraries were prepared using 10X Genomics Chromium and 3’ gene expression kit v3.1. Data processing and analysis was done using 10X Genomics’ Cell Ranger pipeline, Seurat, and consensus non-negative matrix factorization. Results: Using Seurat FindNeighbors, cells in the aggregated dataset were classified into 17 distinct clusters. All clusters were comprised of cells from multiple sites (primary tumor, CTCs, metastases), but three clusters were enriched in CTCs and one cluster was composed of mostly primary tumor cells. All clusters exhibited an epithelial-like gene expression signature score, suggesting that CTC shedding was occurring without prominent epithelial-mesenchymal transition. Consistent with expected differences in oxygenation states, CTC-enriched clusters exhibited a lower hypoxia gene expression score than primary tumor and metastasis-enriched clusters. CTC-enriched clusters also showed higher expression of oxidative phosphorylation genes, suggesting metabolic differences between CTCs and cells from primary tumors and metastases. Based on Human Primary Cell Atlas phenotype prediction, several clusters were associated with stem cell like phenotypes. Additionally, two of three CTC-enriched clusters had elevated expression of mitosis-associated genes, suggesting that at least some populations of CTCs are not quiescent but actively cycling. Conclusions: Utilizing single cell gene expression profiling, we have linked the gene expression profile of CTCs to specific cell subpopulations in primary tumors and metastases. We show that CTC-enriched cell clusters appear to maintain an epithelial phenotype. Subpopulations of CTC cells exhibited enrichment of stemness-associated transcripts and features of active cell cycling. Citation Format: Tomas Vilimas, Brandie Fullmer, Alyssa Chapman, Li Chen, Ting-Chia Chang, Rini Pauly, Biswajit Das, Chris Karlovich, Yvonne A. Evrard, Howard Stotler, Michelle M. Gottholm-Ahalt, Tara Grinnage-Pulley, Melinda G. Hollingshead, James H. Doroshow, P. Mickey Williams. Comparative single cell transcriptome profiling of primary tumors, CTCs and metastatic sites from a bladder cancer PDX model [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P097.

Abstract PO-038: LAMC2: New player in stemness and tumor progression in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a devastating and essentially incurable disease, typically characterized by high chemoresistance and metastatic spread attributable to cancer stem cells (CSCs). This subpopulation is critical for tumor initiation and recurrence, but the mechanism through which they acquire metastatic traits is not well understood. Hence, targeting the CSC niche and their plasticity could be a complementary therapeutic strategy against cancer. Laminin subunit-γ-2 (LAMC2) is an epithelial basement membrane protein, which controls cell motility and adhesion and is widely expressed in the majority of human tumors. However, its role in PDAC remains largely unknown. In several patient cohorts we observed that high levels of LAMC2 significantly correlated with shorter overall survival. In addition, the tissue microarray analysis on PDAC sections revealed prognostic significance of LAMC2 expression in tumor with high grade of aggressiveness (i.e., G2 and G3). To determine the role of LAMC2 in sustaining tumorigenicity, we knocked down it in patient-derived xenografts (PDX) cells using lentiviral shRNA constructs. The silencing of LAMC2 resulted in decreased self-renewal, invasiveness, tumorigenicity and gemcitabine resistance both in vitro and in vivo. To identify or track the LAMC2 tumor cell population in an intact environment we engineered primary PDAC cells that carry EGFP cassette knocked in the LAMC2 locus through the CRISPR-Cas9 technique. Analysis of LAMC2-EGFP+ cells isolated from tumor demonstrated that these cells express a gene program similar to that of highly metastatic stem cells and that they initiate and propagate both the primary tumor and the metastasis to recipient mice very efficiently compared to their counterpart. In conclusion, we identified a highly metastatic subpopulation of cancer stem cells, characterized by high levels of LAMC2. Strategies aimed at targeting the LAMC2 population may be effective in reducing tumor aggressiveness in combination with conventional therapy. Citation Format: Donatella Delle Cave, Tea Teresa Iavazzo, Maria Mangini, Gennaro Andolfi, Teresa Pirozzi, Annalisa Di Domenico, Annachiara De Luca, Enza Lonardo. LAMC2: New player in stemness and tumor progression in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-038.

Cancer Stem Cell Markers, CD44 and ALDH1, for Assessment of Cancer Risk in OPMDs and Lymph Node Metastasis in Oral Squamous Cell Carcinoma.

Tumour heterogeneity in oral cancer is attributed to the presence of cancer stem cells (CSCs). CSCs are the most migratory and metastatic cellular subpopulation within tumours. Assessment of CSC markers as significant predictors of lymph node metastasis may prove valuable in the clinical setting. Furthermore, analysis of this panel of putative stem cell markers in oral dysplasia may additionally inform of the likelihood for oral potentially malignant disorders (OPMDs) to progress to oral squamous cell carcinoma (OSCC). The present study aims to assess the significance of CSC markers in the progression of OPMDs to OSCC and assessment of lymph node metastasis in OSCC. CD44 and ALDH1 were assessed immunohistochemically in 25 normal, 30 OPMDs, and 24 OSCCs. CD44 is a membranous marker and ALDH1 is a cytoplasmic marker. The immunohistochemical expression of these markers were compared between OPMDs with and without dysplasia, as well as between low-risk and high-risk dysplasias. Similarly, expression was compared between OSCC with and without lymph node metastasis and among grades of OSCC. Positive CD44 expression was seen in all normal mucosal tissues. The expression decreased from normal epithelium to OPMDs but increased in OSCC. CD44 expression was positive in 21 cases of OSCC (87.5%) and reduced from well-differentiated to poorly differentiated OSCC. CD44 staining index was higher in OSCC without lymph node metastasis (3.59) when compared with OSCC with lymph node metastasis (1.33). There was a statistically significant difference observed in the ALDH1 staining index among three groups (p < 0.05), with highest expression seen in OSCC. Within OPMDs, the ALDH1 staining index was statistically higher in OPMDs with dysplasia as compared to OPMDs without dysplasia. Furthermore, the expression was higher in OPMDs with high-risk dysplasia when compared with low-risk dysplasia, but this was not statistically significant (p = 0.82). In conclusion, The CD44 positive population possesses properties of CSCs in head and neck carcinoma, and continuous shedding could be found after CD44 down-regulation. The present study reports differences in ALDH1 expression between OPMDs with and without dysplasia, dysplastic and non-dysplastic epithelia, and low-risk and high-risk dysplasia. These findings may suggest ALDH1 as a specific marker for dysplasia. CD44 demonstrated a difference in staining index in OSCC without lymph node metastasis versus OSCC with lymph node metastasis. These findings may suggest CD44 as a marker for lymph node metastasis. Both proteins may play key roles in the tumorigenicity of CSCs in OPMDs and OSCC.

Glutathione peroxidase 4-dependent glutathione high-consumption drives acquired platinum chemoresistance in lung cancer-derived brain metastasis.

BackgroundPlatinum‐based chemotherapy is effective in inducing shrinkage of primary lung cancer lesions; however, it shows finite therapeutic efficacy in patients suffering from brain metastasis (BM). The intrinsic changes of BM cells, which contribute to the poor results remain unknown.MethodsPlatinum drug‐sensitivity was assessed by utilizing a preclinical BM model of PC9 lung adenocarcinoma cells in vitro and in vivo. High consumption of glutathione (GSH) and two associated upregulated proteins (GPX4 and GSTM1) in BM were identified by integrated metabolomics and proteomics in cell lines and verified by clinical serum sample. Gain‐of‐function and rescue experiments were implemented to reveal the impact and mechanism of GPX4 and GSTM1 on the chemosensitivity in BM. The interaction between GPX4 and GSTM1 was examined by immunoblotting and immunoprecipitation. The mechanism of upregulation of GPX4 was further uncovered by luciferase reporter assay, immunoprecipitation, and electrophoretic mobility shift assay.ResultsThe derivative brain metastatic subpopulations (PC9‐BrMs) of parental cells PC9 developed obvious resistance to platinum. Radically altered profiles of BM metabolism and protein expression compared with primary lung cancer cells were described and GPX4 and GSTM1 were identified as being responsible for the high consumption of GSH, leading to decreased chemosensitivity by negatively regulating ferroptosis. Besides, GSTM1 was found regulated by GPX4, which was transcriptionally activated by the Wnt/NR2F2 signaling axis in BM.ConclusionsCollectively, our findings demonstrated that Wnt/NR2F2/GPX4 promoted acquired chemoresistance by suppressing ferroptosis with high consumption of GSH. GPX4 inhibitor was found to augment the anticancer effect of platinum drugs in lung cancer BM, providing novel strategies for lung cancer patients with BM.

Single-cell lineage tracing of metastatic cancer reveals selection of hybrid EMT states.

The underpinnings of cancer metastasis remain poorly understood, in part due to a lack of tools for probing their emergence at high resolution. Here we present macsGESTALT, an inducible CRISPR-Cas9-based lineage recorder with highly efficient single-cell capture of both transcriptional and phylogenetic information. Applying macsGESTALT to a mouse model of metastatic pancreatic cancer, we recover ∼380,000 CRISPR target sites and reconstruct dissemination of ∼28,000 single cells across multiple metastatic sites. We find that cells occupy a continuum of epithelial-to-mesenchymal transition (EMT) states. Metastatic potential peaks in rare, late-hybrid EMT states, which are aggressively selected from a predominately epithelial ancestral pool. The gene signatures of these late-hybrid EMT states are predictive of reduced survival in both human pancreatic and lung cancer patients, highlighting their relevance to clinical disease progression. Finally, we observe evidence for invivo propagation of S100 family gene expression across clonally distinct metastatic subpopulations.

Phenotypic Heterogeneity and Metastasis of Breast Cancer Cells.

Although intratumoral genomic heterogeneity can impede cancer research and treatment, less is known about the effects of phenotypic heterogeneities. To investigate the role of cell migration heterogeneities in metastasis, we phenotypically sorted metastatic breast cancer cells into two subpopulations based on migration ability. Although migration is typically considered to be associated with metastasis, when injected orthotopically in vivo, the weakly migratory subpopulation metastasized significantly more than the highly migratory subpopulation. To investigate the mechanism behind this observation, both subpopulations were assessed at each stage of the metastatic cascade, including dissemination from the primary tumor, survival in the circulation, extravasation, and colonization. Although both subpopulations performed each step successfully, weakly migratory cells presented as circulating tumor cell (CTC) clusters in the circulation, suggesting clustering as one potential mechanism behind the increased metastasis of weakly migratory cells. RNA sequencing revealed weakly migratory subpopulations to be more epithelial and highly migratory subpopulations to be more mesenchymal. Depletion of E-cadherin expression from weakly migratory cells abrogated metastasis. Conversely, induction of E-cadherin expression in highly migratory cells increased metastasis. Clinical patient data and blood samples showed that CTC clustering and E-cadherin expression are both associated with worsened patient outcome. This study demonstrates that deconvolving phenotypic heterogeneities can reveal fundamental insights into metastatic progression. More specifically, these results indicate that migratory ability does not necessarily correlate with metastatic potential and that E-cadherin promotes metastasis in phenotypically sorted breast cancer cell subpopulations by enabling CTC clustering. SIGNIFICANCE: This study employs phenotypic cell sorting for migration to reveal a weakly migratory, highly metastatic breast cancer cell subpopulation regulated by E-cadherin, highlighting the dichotomy between cancer cell migration and metastasis.

Development of a highly pulmonary metastatic orthotopic renal cell carcinoma murine model.

ABSTRACTThe incidence of renal cell carcinoma (RCC) is high, and its outcomes remain poor. Mortality is attributable largely to metastatic disease and a dearth of effective therapeutic interventions. The lungs are the most common metastatic site. To elucidate the biological mechanisms underlying pulmonary metastasis and identify superior therapeutic strategies, we developed a novel and clinically relevant murine RCC model exhibiting enhanced pulmonary metastasis. Mice underwent intrarenal implantation using luciferase-expressing Renca, a murine renal adenocarcinoma cell line. Primary renal tumor progression and development of metastatic lung lesions were monitored in live mice using bioluminescent imaging, followed by post-mortem organ assessment. Cells were isolated from pulmonary metastases for reimplantation, followed by repeat monitoring and assessment. This process was repeated once more for a total of two in vivo passages to select for pulmonary metastatic Renca cell subpopulations. However, a single round of in vivo selection was sufficient to produce a near-maximally metastatic subpopulation. Relative to Renca cell-implanted mice, subpopulation-implanted mice exhibited shorter implantation-metastasis intervals (5 days), shorter implantation-moribundity intervals (sacrificed at 18.6±2.9 versus 22.3±1.1 days), a higher number of metastatic lung lesions at 23 days (183.9±39.0 versus 172.6±38.2) and poorer survival. Implantation of cells derived from the second round of in vivo selection produced no further significant differences in the above metrics. This model consistently and efficiently recapitulates RCC pulmonary metastasis while allowing in vivo monitoring of tumor progression, thereby facilitating elucidation of the molecular mechanisms underlying pulmonary metastasis and evaluation of therapeutic modalities.

N6 -Methyladenosine Regulates mRNA Stability and Translation Efficiency of KRT7 to Promote Breast Cancer Lung Metastasis.

The roles of RNA modification during organ metastasis of cancer cells are not known. Here we established breast cancer lung metastasis cells by three rounds of selection of lung metastatic subpopulations in vivo and designated them as BCLMF3 cells. In these cells, mRNA N6 -methyladenosine (m6A) and methyltransferase METTL3 were increased, while the demethylase FTO was decreased. Epi-transcriptome and transcriptome analyses together with functional studies identified keratin 7 (KRT7) as a key effector for m6A-induced breast cancer lung metastasis. Specifically, increased METTL3 methylated KRT7-AS at A877 to increase the stability of a KRT7-AS/KRT7 mRNA duplex via IGF2BP1/HuR complexes. Furthermore, YTHDF1/eEF-1 was involved in FTO-regulated translational elongation of KRT7 mRNA, with methylated A950 in KRT7 exon 6 as the key site for methylation. In vivo and clinical studies confirmed the essential roles of KRT7, KRT7-AS, and METTL3 for lung metastasis and clinical progression of breast cancer. Collectively, m6A promotes breast cancer lung metastasis by increasing the stability of a KRT7-AS/KRT7 mRNA duplex and translation of KRT7. SIGNIFICANCE: This study suggests that N6 -methyladenosine is a key driver and potential therapeutic target in breast cancer metastasis.

Abstract B72: Dissecting the heterogeneity of metastatic neuroblastoma cells by single-cell RNA-seq

Abstract Introduction: The major cause of death in neuroblastoma (NB) is metastatic relapse, and most therapies do not specifically target metastases. We previously developed a metastatic mouse model using intracardiac injection of tagged human SK-N-AS NB cells, followed by multiple rounds of in vivo selection from bone and brain metastatic sites. Gene expression profiles identified novel genes and pathways dysregulated in metastatic NB cells that, when genetically or pharmacologically manipulated, affected metastasis. A subset of genes up- or downregulated in the enhanced metastatic cells was used to generate a metastatic gene signature that predicted patient survival. We have now asked (1) whether there is a transcriptionally distinct subpopulation of cells in the parental population that selectively metastasizes to bone and brain, and (2) if there are transcriptional differences that can account for the ability of the enhanced metastatic cells to hone to bone/bone marrow vs. brain. Methods: We used 10X Genomics single-cell RNA sequencing (scRNA-seq) to transcriptionally profile parental cells and two in vivo-selected cell lines that showed enhanced metastatic spread to bone/bone marrow (B5) and brain (BR2). Approximately 5,000 genes and 10,000 transcripts per cell were sequenced, with the data analyzed using a unique analysis pipeline incorporating extensive data quality analysis with visualization and clustering methods using evidence-based parameter selection. Genes with high variance were used to compute principal components as inputs to visualize cells in two dimensions (t-SNE plots) in clusters. Results: scRNA-seq data showed that the parental cells clustered separately from B5 and BR2 cells, with clusters in the parental cells that expressed genes enriched in the B5 and BR2 cells that we previously reported drove migration/invasion in vitro and/or metastasis in vivo, including sphingosine kinase 1. These results suggest that subpopulations of the parental cells with enhanced metastatic gene expression are selected in vivo for honing to metastatic sites. We identified genes enriched in B5 and BR2 cells encoding cell surface proteins, chemokines including CCL2, and transcription factors including Msx1 involved in metastasis of other cancers, that we will assess for roles in NB metastasis. While the B2 and BR5 cells were transcriptionally similar to each other, we did identify genes highly expressed in B2 subclusters that are associated with bone cancers including FGF5, and that are highly expressed in BR2 subclusters that are associated with brain cancers including RARRES2. These findings suggest that transcriptional differences between B5 and BR2 cells may influence metastasis to the bone or brain. Conclusion: The identification of genes that are differentially expressed in the more metastatic subpopulations will contribute to our understanding of the molecular mechanisms governing metastases and identify novel targetable pathways that were not detected by profiling bulk tumor populations. Citation Format: Alice R. Shan, Alexander Gont, David R. Kaplan, Meredith S. Irwin. Dissecting the heterogeneity of metastatic neuroblastoma cells by single-cell RNA-seq [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B72.