Significance Of Metastasis Research Articles

RBFOX2 modulates a metastatic signature of alternative splicing in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDA) is characterized by aggressive local invasion and metastatic spread, leading to high lethality. Although driver gene mutations during PDA progression are conserved, no specific mutation is correlated with the dissemination of metastases1–3. Here we analysed RNA splicing data of a large cohort of primary and metastatic PDA tumours to identify differentially spliced events that correlate with PDA progression. De novo motif analysis of these events detected enrichment of motifs with high similarity to the RBFOX2 motif. Overexpression of RBFOX2 in a patient-derived xenograft (PDX) metastatic PDA cell line drastically reduced the metastatic potential of these cells in vitro and in vivo, whereas depletion of RBFOX2 in primary pancreatic tumour cell lines increased the metastatic potential of these cells. These findings support the role of RBFOX2 as a potent metastatic suppressor in PDA. RNA-sequencing and splicing analysis of RBFOX2 target genes revealed enrichment of genes in the RHO GTPase pathways, suggesting a role of RBFOX2 splicing activity in cytoskeletal organization and focal adhesion formation. Modulation of RBFOX2-regulated splicing events, such as via myosin phosphatase RHO-interacting protein (MPRIP), is associated with PDA metastases, altered cytoskeletal organization and the induction of focal adhesion formation. Our results implicate the splicing-regulatory function of RBFOX2 as a tumour suppressor in PDA and suggest a therapeutic approach for metastatic PDA.

Identification of lymph node metastasis-related genes and patterns of immune infiltration in colon adenocarcinoma.

Colon adenocarcinoma(COAD) is one of the most common tumors of the digestive tract. Lymph node metastasis (LNM) is a well-established prognostic factor for COAD. The mechanism of COAD lymph node metastasis in immunology remains unknown. The identification of LNM-related biomarkers of COAD could help in its treatment. Thus, the current study was aimed to identify key genes and construct a prognostic signature. Gene expression and clinical data were obtained from The Cancer Genome Atlas (TCGA) database. Differentially expressed genes were calculated by using R software. GO functional and KEGG pathway enrichment analysis were processed. The CIBERSORT algorithm was used to assess immune cell infiltration. STRING database was used to screen key genes and constructed a protein-protein interaction network (PPI network). The LASSO-Cox regression analysis was performed based on the components of the PPI network. The correlation analysis between LNM-related signature and immune infiltrating cells was then investigated. TISIDB was used to explore the correlation between the abundance of immunomodulators and the expression of the inquired gene. In total, 394 differentially expressed genes were identified. After constructing and analyzing the PPI network, 180 genes were entered into the LASSO-Cox regression model, constructing a gene signature. Five genes(PMCH, LRP2, NAT1, NKAIN4, and CD1B) were identified as LNM-related genes of clinical value. Correlation analysis revealed that LRP2 and T follicular helper cells (R=0.34, P=0.0019) and NKAIN4 and T follicular helper cells (R=0.23, P=0.041) had significant correlations. Immunologic analysis revealed that LRP2 and NKAIN4 are potential coregulators of immune checkpoints in COAD. In general, this study revealed the key genes related to lymph node metastasis and prognostic signature. Several potential mechanisms and therapeutic and prognostic targets of lymph node metastasis were also demonstrated in COAD.

Abstract PR007: Comprehensive ctDNA profiling reveals potential metastatic genomic signatures in treatment-naive early-stage breast cancer patients

Abstract Background: Genomic profiling has revolutionized precision oncology impacting the diagnosis, prognosis, and therapy decisions. Considering high spatiotemporal diversity and heterogenicity of breast tumor-cell genomes, small-gene panels often fail to capture rare but important genomic alterations. Conversely, comprehensive ctDNA sequencing approaches enable the identification of under characterized ‘long tailed driver’ genomic alterations and capture Intra and inter metastatic heterogeneity. Here, we demonstrate the clinical utility of comprehensive genome profiling with higher sensitivity to predict the possibility of metastasis in early-stage breast cancer patients. Methods: We retrospectively analyzed ctDNA and genomic DNA (gDNA) from FFPE samples as well as circulating tumor cells (CTC) in 10 treatment-naive hormone positive and HER2 negative, primary-stage breast cancer patients [GS1] using the OncoIndx comprehensive 600 gene panel. The panel captures all important cancer-relevant genomic alterations including Tumor Mutation Burden (TMB), Micro Satellite Instability (MSI), homologous recombination deficiency (HRD) prediction, and cfDNA tumor fraction (TF). CTCs were enumerated from 1.5 ml of blood using the OncoDiscover platform approved by the Drug Controller General of India having anti-EpCAM antibody-mediated immunomagnetic nanoparticles. CTCs were confirmed for cytokeratin 18+ and DAPI + markers and the absence of CD45. Results: Comprehensive genomic profile obtained from ctDNA and gDNA from FFPE of early-stage breast cancer patients predominantly exhibited the presence of alterations in PIK3CA and ESR1 signaling pathways. PIK3CA mutations were present in 77% and 44% of baseline ctDNA and gDNA samples, while ESR1 mutations were present in 44% and 22% of baseline ctDNA and gDNA, respectively. In addition, we observed about 70% additional driver mutations in ctDNA samples suggesting shedding of ctDNA together with CTC (80% positive), a likely positive biomarker of metastasis. About 50% of the patients showed higher TMB and HRD. Notably, TF representing ctDNA varied between 13% to 27% in blood samples with a corresponding ploidy range of 2.9 to 4.7. Surprisingly, ~50% of the patient population matched the mutation profile of clinically confirmed metastatic patients. All the patients harboring potential metastatic driver alterations showed the presence of CTCs in peripheral blood. Conclusions: Comprehensive ctDNA genomic profiling showed potential metastasis driving alterations suggesting the role of ctDNA-based liquid biopsy to predict metastasis in early breast cancer patients. We observed enhanced TF at the time of diagnosis, possibly due to the presence of distant metastasis, high disease burden, and aggressive tumor biology. Our results suggest that ctDNA dynamics at the time of disease presentation can predict early metastasis, and may demonstrate the divergent response of tumor heterogeneity to treatment in early-stage breast cancer. Citation Format: Gowhar Shafi, Manoj Dongare, Atul Bharde, Moubeen Fauzul, Kanchan Hariramani, Alain D’Souza, Bhagwat Jadhav, Trupti Kad, Sangeeta Prajapati, Vikas Jadhav, ManojKumar Kumaran, Sumit Haldar, Vatsal Mehra, Sujit Joshi, Gourishankar Aland, Richa Dave, Sreeja Jayant, Aravindan Vasudevan, Mohan Uttarwar, Jayant Khandare. Comprehensive ctDNA profiling reveals potential metastatic genomic signatures in treatment-naive early-stage breast cancer patients [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 PR007.

Preclinical Evaluation of a Novel Small Molecule LCC-21 to Suppress Colorectal Cancer Malignancy by Inhibiting Angiogenic and Metastatic Signatures.

Colorectal cancer (CRC) is one of the most common cancers, and it frequently metastasizes to the liver and lymph nodes. Despite major advances in treatment modalities, CRC remains a poorly characterized biological malignancy, with high reported cases of deaths globally. Moreover, cancer stem cells (CSCs) and their microenvironment have been widely shown to promote colon cancer development, progression, and metastasis. Therefore, an understanding of the underlying mechanisms that contribute to the maintenance of CSCs and their markers in CRC is crucial in efforts to treat cancer metastasis and develop specific therapeutic targets for augmenting current standard treatments. Herein, we applied computational simulations using bioinformatics to identify potential theranostic markers for CRC. We identified the overexpression of vascular endothelial growth factor-α (VEGFA)/β-catenin/matrix metalloproteinase (MMP)-7/Cluster of Differentiation 44 (CD44) in CRC to be associated with cancer progression, stemness, resistance to therapy, metastasis, and poor clinical outcomes. To further investigate, we explored in silico molecular docking, which revealed potential inhibitory activities of LCC-21 as a potential multitarget small molecule for VEGF-A/CTNNB1/MMP7/CD44 oncogenic signatures, with the highest binding affinities displayed. We validated these finding in vitro and demonstrated that LCC-21 inhibited colony and sphere formation, migration, and invasion, and these results were further confirmed by a Western blot analysis in HCT116 and DLD-1 cells. Thus, the inhibitory effects of LCC-21 on these angiogenic and onco-immunogenic signatures could be of translational relevance as potential CRC biomarkers for early diagnosis.

Highlights from Recent Cancer Literature.

Highlights from Recent Cancer Literature.

Estimating Metastatic Risk of Pancreatic Ductal Adenocarcinoma at Single-Cell Resolution

Pancreatic ductal adenocarcinoma (PDAC) is characterized by intra-tumoral heterogeneity, and patients are always diagnosed after metastasis. Thus, finding out how to effectively estimate metastatic risk underlying PDAC is necessary. In this study, we proposed scMetR to evaluate the metastatic risk of tumor cells based on single-cell RNA sequencing (scRNA-seq) data. First, we identified diverse cell types, including tumor cells and other cell types. Next, we grouped tumor cells into three sub-populations according to scMetR score, including metastasis-featuring tumor cells (MFTC), transitional metastatic tumor cells (TransMTC), and conventional tumor cells (ConvTC). We identified metastatic signature genes (MSGs) through comparing MFTC and ConvTC. Functional enrichment analysis showed that up-regulated MSGs were enriched in multiple metastasis-associated pathways. We also found that patients with high expression of up-regulated MSGs had worse prognosis. Spatial mapping of MFTC showed that they are preferentially located in the cancer and duct epithelium region, which was enriched with the ductal cells' associated inflammation. Further, we inferred cell-cell interactions, and observed that interactions of the ADGRE5 signaling pathway, which is associated with metastasis, were increased in MFTC compared to other tumor sub-populations. Finally, we predicted 12 candidate drugs that had the potential to reverse expression of MSGs. Taken together, we have proposed scMetR to estimate metastatic risk in PDAC patients at single-cell resolution which might facilitate the dissection of tumor heterogeneity.

One Step Nucleic Acid Amplification (OSNA) Lysate Samples Are Suitable to Establish a Transcriptional Metastatic Signature in Patients with Early Stage Hormone Receptors-Positive Breast Cancer.

The One Step Nucleic Acid Amplification (OSNA) is being adopted worldwide for sentinel lymph nodes (SLNs) staging in breast cancer (BC). As major disadvantage, OSNA precludes prognostic information based on structural evaluation of SLNs. Our aim is to identify biomarkers related to tumor-microenvironment interplay exploring gene expression data from the OSNA remaining lysate. This study included 32 patients with early stage hormone receptors-positive BC. Remaining OSNA lysates were prepared for targeted RNA-sequencing analysis. Identification of differentially expressed genes (DEGs) was performed by DESeq2 in R and data analysis in STATA. The results show that, in metastatic SLNs, several genes were upregulated: KRT7, VTCN1, CD44, GATA3, ALOX15B, RORC, NECTIN2, LRG1, CD276, FOXM1 and IGF1R. Hierarchical clustering analysis revealed three different clusters. The identified DEGs codify proteins mainly involved in cancer aggressiveness and with impact in immune response. The overexpression of the immune suppressive genes VTCN1 and CD276 may explain that no direct evidence of activation of immune response in metastatic SLNs was found. We show that OSNA results may be improved incorporating microenvironment-related biomarkers that may be useful in the future for prognosis stratification and immunotherapy selection. As OSNA assay is being implemented for SLNs staging in other cancers, this approach could also have a wider utility.

Stratification of lymph node metastases as macrometastases, micrometastases, or isolated tumor cells has no clinical implication in patients with cervical cancer: Subgroup analysis of the SCCAN project

BackgroundIn cervical cancer, presence of lymph-node macrometastases (MAC) is a major prognostic factor and an indication for adjuvant treatment. However, since clinical impact of micrometastases (MIC) and isolated tumor-cells (ITC) remains controversial, we sought to identify a cut-off value for the metastasis size not associated with negative prognosis. MethodsWe analyzed data from 967 cervical cancer patients (T1a1L1-T2b) registered in the SCCAN (Surveillance in Cervical CANcer) database, who underwent primary surgical treatment, including sentinel lymph-node (SLN) biopsy with pathological ultrastaging. The size of SLN metastasis was considered a continuous variable and multiple testing was performed for cut-off values of 0.01–1.0 mm. Disease-free survival (DFS) was compared between N0 and subgroups of N1 patients defined by cut-off ranges. ResultsLN metastases were found in 172 (18%) patients, classified as MAC, MIC, and ITC in 79, 54, and 39 patients, respectively. DFS was shorter in patients with MAC (HR 2.20, P = 0.003) and MIC (HR 2.87, P < 0.001), while not differing between MAC/MIC (P = 0.484). DFS in the ITC subgroup was neither different from N0 (P = 0.127) nor from MIC/MAC subgroups (P = 0.449). Cut-off analysis revealed significantly shorter DFS compared to N0 in all subgroups with metastases ≥0.4 mm (HR 2.311, P = 0.04). The significance of metastases <0.4 mm could not be assessed due to limited statistical power (<80%). We did not identify any cut-off for the size of metastasis with significantly better prognosis than the rest of N1 group. ConclusionsIn cervical cancer patients, the presence of LN metastases ≥0.4 mm was associated with a significant negative impact on DFS and no cut-off value for the size of metastasis with better prognosis than N1 was found. Traditional metastasis stratification based on size has no clinical implication.

Highly expressed FAM189B predicts poor prognosis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most malignant tumors with persistently high morbidity and mortality. However, the expression, prognostic and clinical significance of FAM189 family genes in HCC remain largely unknown. In this study, the expression levels of FAM189 family genes in HCC were analyzed through TCGA-LIHC and ICGC-LIRI-JP cohorts, and further validated in multiple independent GEO datasets. It was found that the expression of FAM189B was significantly upregulated in HCC tumor tissues, while the expression of FAM189A1 and FAM189A2 was not significantly changed between tumor and adjacent tissues. Further analysis revealed that upregulated copy number variation contributed to increased expression of FAM189B in HCC. Survival analysis showed that highly expressed FAM189B was significantly correlated with unfavorable prognosis, including overall survival, disease-specific survival, and progression-free interval. Univariate and multivariate Cox regression analysis showed that FAM189B was a potential novel prognosis factor for HCC patients. In addition, the association between FAM189B expression and clinical and molecular characteristics was analyzed. High expression of FAM189B was associated with high AFP level, high predicted risk metastasis signature, and TP53 mutation, while there was no significant association between FAM189B expression and cancer stage or tumor grade of HCC. Gene set enrichment analysis revealed that highly expressed FAM189B was closely related with signal pathways and biological processes associated with cell proliferation and cell cycle in HCC. In conclusion, this study suggested that FAM189B was highly expressed in HCC and highly expressed FAM189B may serve as an effective prognostic indicator and a potential therapeutic target for HCC patients.

Connecting multiple microenvironment proteomes uncovers the biology in head and neck cancer

The poor prognosis of head and neck cancer (HNC) is associated with metastasis within the lymph nodes (LNs). Herein, the proteome of 140 multisite samples from a 59-HNC patient cohort, including primary and matched LN-negative or -positive tissues, saliva, and blood cells, reveals insights into the biology and potential metastasis biomarkers that may assist in clinical decision-making. Protein profiles are strictly associated with immune modulation across datasets, and this provides the basis for investigating immune markers associated with metastasis. The proteome of LN metastatic cells recapitulates the proteome of the primary tumor sites. Conversely, the LN microenvironment proteome highlights the candidate prognostic markers. By integrating prioritized peptide, protein, and transcript levels with machine learning models, we identify nodal metastasis signatures in blood and saliva. We present a proteomic characterization wiring multiple sites in HNC, thus providing a promising basis for understanding tumoral biology and identifying metastasis-associated signatures.

CEBPB is associated with active tumor immune environment and favorable prognosis of metastatic skin cutaneous melanoma.

Metastatic skin cutaneous melanoma (SKCM) is a common malignancy that accounts for low morbidity but high mortality of skin cancer. SKCM is characterized by high lymphocytic infiltration, whereas the states of infiltrated cells are variable in patients leading to a heterogeneous prognosis and hindering appropriate clinical decisions. It is therefore urgent to identify markers associated with lymphocytic infiltration, cellular conditions, and the prognosis of SKCM. In this study, we report that CEBPB, a transcriptional factor, is mainly expressed in macrophages in metastatic SKCM and associated with an active tumor immune environment and a favorable prognosis through integrated analysis of single-cell and bulk RNA-seq datasets. High CEBPB expression is significantly associated with active inflammation and immune response pathways in both macrophages and bulk SKCM tumor tissues. A signature based on CEBPB-associated genes that are specifically expressed in macrophages could robustly and prognostically separate different metastatic SKCM patients. In addition, the associations between the metastatic SKCM tumor signature and microenvironment with respect to T-cell recruitment and state, inflammation response, angiogenesis, and so on were also determined. In conclusion, we present here the first report on CEBPB in tumor immune environment and prognosis regulation in metastatic SKCM and construct a reliable signature, which should provide a useful biomarker for stratification of the patient's prognosis and therapeutic selection.

A transcriptional metastatic signature predicts survival in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer in adults. When ccRCC is localized to the kidney, surgical resection or ablation of the tumor is often curative. However, in the metastatic setting, ccRCC remains a highly lethal disease. Here we use fresh patient samples that include treatment-naive primary tumor tissue, matched adjacent normal kidney tissue, as well as tumor samples collected from patients with bone metastases. Single-cell transcriptomic analysis of tumor cells from the primary tumors reveals a distinct transcriptional signature that is predictive of metastatic potential and patient survival. Analysis of supporting stromal cells within the tumor environment demonstrates vascular remodeling within the endothelial cells. An in silico cell-to-cell interaction analysis highlights the CXCL9/CXCL10-CXCR3 axis and the CD70-CD27 axis as potential therapeutic targets. Our findings provide biological insights into the interplay between tumor cells and the ccRCC microenvironment.

A transcriptional metastatic signature predicts survival in clear cell renal cell carcinoma

A transcriptional metastatic signature predicts survival in clear cell renal cell carcinoma

HER2 drives lung fibrosis by activating a metastatic cancer signature in invasive lung fibroblasts.

Progressive tissue fibrosis, including idiopathic pulmonary fibrosis (IPF), is characterized by excessive recruitment of fibroblasts to sites of tissue injury and unremitting extracellular matrix deposition associated with severe morbidity and mortality. However, the molecular mechanisms that control progressive IPF have yet to be fully determined. Previous studies suggested that invasive fibroblasts drive disease progression in IPF. Here, we report profiling of invasive and noninvasive fibroblasts from IPF patients and healthy donors. Pathway analysis revealed that the activated signatures of the invasive fibroblasts, the top of which was ERBB2 (HER2), showed great similarities to those of metastatic lung adenocarcinoma cancer cells. Activation of HER2 in normal lung fibroblasts led to a more invasive genetic program and worsened fibroblast invasion and lung fibrosis, while antagonizing HER2 signaling blunted fibroblast invasion and ameliorated lung fibrosis. These findings suggest that HER2 signaling may be a key driver of fibroblast invasion and serve as an attractive target for therapeutic intervention in IPF.

Definition and verification of novel metastasis and recurrence related signatures of ccRCC: A multicohort study.

Cancer metastasis and recurrence remain major challenges in renal carcinoma patient management. There are limited biomarkers to predict the metastatic probability of renal cancer, especially in the early-stage subgroup. Here, our study applied robust machine-learning algorithms to identify metastatic and recurrence-related signatures across multiple renal cancer cohorts, which reached high accuracy in both training and testing cohorts. Clear cell renal cell carcinoma (ccRCC) patients with primary or metastatic site sequencing information from eight cohorts, including one out-house cohort, were enrolled in this study. Three robust machine-learning algorithms were applied to identify metastatic signatures. Then, two distinct metastatic-related subtypes were identified and verified; matrix remodeling associated 5(MXRA5), as a promising diagnostic and therapeutic target, was investigated in vivo and in vitro. We identified five stable metastasis-related signatures (renin, integrin subunit beta-like 1, MXRA5, mesenchyme homeobox 2, and anoctamin 3) from multicenter cohorts. Additionally, we verified the specificity and sensibility of these signatures in external and out-house cohorts, which displayed a satisfactory consistency. According to these metastatic signatures, patients were grouped into two distinct and heterogeneous ccRCC subtypes named metastatic cancer subtype 1 (MTCS1) and type 2 (MTCS2). MTCS2 exhibited poorer clinical outcomes and metastatic tendencies than MTCS1. In addition, MTCS2 showed higher immune cell infiltration and immune signature expression but a lower response rate to immune blockade therapy than MTCS1. The MTCS2 subgroup was more sensitive to saracatinib, sunitinib, and several molecular targeted drugs. In addition, MTCS2 displayed a higher genome mutation burden and instability. Furthermore, we constructed a prognosis model based on subtype biomarkers, which performed well in training and validation cohorts. Finally, MXRA5, as a promising biomarker, significantly suppressed malignant ability, including the cell migration and proliferation of ccRCC cell lines in vitro and in vivo. This study identified five robust metastatic signatures and proposed two metastatic probability clusters with stratified prognoses, multiomics landscapes, and treatment options. The current work not only provided new insight into the heterogeneity of renal cancer but also shed light on optimizing decision-making in immunotherapy and chemotherapy.

Signature and Prediction of Perigastric Lymph Node Metastasis in Patients with Gastric Cancer and Total Gastrectomy: Is Total Gastrectomy Always Necessary?

Simple SummaryThe signature and prediction of perigastric lymph node metastasis (pLNM) is clinically important, but evidence is still lacking. Here, we aimed to identify an informative signature for the prediction of pLNMs in gastric cancer patients after total gastrectomy, and reassess the current indications for proximal gastrectomy and pylorus-preserving gastrectomy (PPG). We found that proximal gastrectomy may be expanded to patients with stage T1–T2 GC and/or tumor diameter < 4 cm in the upper-third stomach, while PPG may be expanded to include T1–T2/N0 and/or tumors < 4 cm in the middle-third stomach. Furthermore, we developed a new predictive factor, the shortest distance from the pylorus ring to the distal edge of the tumor, which showed good predictive performance for pLNMs.Background: A growing number of studies suggest that the current indications for partial gastrectomy, including proximal gastrectomy and pylorus-preserving gastrectomy (PPG), may be expanded, but evidence is still lacking. Methods: We retrospectively analyzed 300 patients with gastric cancer (GC) who underwent total gastrectomy. We analyzed the incidence of pLNMs in relation to tumor location, tumor size and T stage. We further identified predictive factors for perigastric lymph node metastasis (pLNM) in stations 1, 2, 3, 4sa, 4sb, 4d, 5, and 6. Results: No patients with upper-third T1–T2 stage GC had pLNMs in stations 4sa, 4sb, 4d, 5, or 6, but 3.8% of patients with stage T3 had 4d pLNM. No patients with upper-third GC < 4 cm in diameter had pLNMs in 2, 4sa, 4d, 5, or 6, and 2.3% of patients had pLNMs in 4sb. For middle-third GCs, 2.9% of patients with T1 stage had pLNMs in 4sa and 5, but no patients with T2 stage or tumors < 4 cm had pLNMs in 2, 4sa, or 5. The shortest distance from pylorus ring to distal edge of tumor (sDPD) was a new predictive factor for pLNMs in 2, 4d, 5, and 6. Conclusions: Proximal gastrectomy may be expanded to patients with stage T1–T2 GC and/or tumor diameter < 4 cm in the upper-third stomach, whereas PPG may be expanded to include T1–T2/N0 and/or tumors < 4 cm in the middle-third stomach. A new predictive factor, sDPD, showed good predictive performance for pLNMs, especially in stations 4d, 5, and 6.

Abstract 982: A new transcriptional metastatic signature predicts survival in clear cell renal cell carcinoma

Abstract Introduction: Renal cell carcinoma, particularly the clear cell histological subtype (ccRCC), is the most common kidney cancer in adults, accounting for 75-85% of all cases. When it is localized to the kidney, it is often cured with surgical resection or thermal ablation. One quarter of the patients have evidence of metastatic disease at the time of diagnosis, and in this setting ccRCC still is a highly lethal disease with few effective treatment options. In this study we used single-cell RNA-sequencing to characterize tumor cell clonality, metastatic signatures and the tumor microenvironment in ccRCC at the single-cell level. Methodological approach: Here we took advantage of fresh patient samples that included treatment-naive primary tumor tissue, matched adjacent normal kidney tissue, as well as tumor samples collected from patients with bone metastases. We used single cell RNA sequencing (10X Genomics, Chromium 3’ v2 kit, 10x Genomics), that was analyzed with PAGODA and CONOS. Summary of new unpublished data: Our analysis revealed a distinct transcriptional signature that predicts metastatic potential and patient survival was disclosed through the single-cell analysis. In-depth exploration of the supporting stromal cells displayed a phenotype of vascular remodeling within the endothelial cells. The fibroblasts showed a proliferative signature that was associated with poor survival. An in silico cell-to-cell interaction analysis highlighted the CXCL9/CXCL10-CXCR3 axis and the CD70-CD27 axis as potential therapeutic targets. Conclusions: Our discoveries in ccRCC provide complementary biological insights, specifically in the interplay between tumor cells and their microenvironment in ccRCC. We demonstrate important biological insights into ccRCC biology including: (1) identification of a distinct metastatic signature that predicts survival outcome, (2) discovery of a proximal tubule cell population that may represent the ccRCC cell of origin, and (3) a detailed exploration in the immunosuppressive environment and the stromal alterations in treatment-naïve patients. In addition, we stress potential therapeutic targets for further evaluation in preclinical models. Citation Format: Adele Mirna Alchahin, Shenglin Mei, Ioanna Tsea, Taghreed Hirz, Youmna Kfoury, Douglas Dahl, Chin-Lee Wu, Alexander O. Subtelny, Shulin WU, David T. Scadden, John H. Shin, Philip J. Saylor, David B. Sykes, Peter V. Kharchenko, Ninib Baryawno. A new transcriptional metastatic signature predicts survival in clear cell renal cell carcinoma [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 982.

Abstract 5982: Single-cell RNA sequencing (scRNA-Seq) from mice model reveals metastatic and stemness signatures in esophageal squamous cell carcinoma (ESCC)

Abstract Remote oligometastases are the predominant roots of the unsatisfactory 5-year survival rate in esophageal squamous cell carcinoma (ESCC) patients. The title of metastatic-initiating cells (MICs) has been designated to describe tumor subpopulation with heterogeneous genetic background and behaviors that potentially displays stemness features and favors metastatic lesion formation. However, preceding studies have overpassed the early surviving MICs due to limitations in bulk transcriptomic sequencing at macrometastatic stage. With the aim of denoting and characterizing MICs in ESCC, we established the pulmonary metastatic mice model via intravenous injection and followed by multiplex single-cell RNA sequencing (scRNA-Seq) of tumor cells collected from lung tissues along longitudinal end-points. The scRNA-Seq results uncover a 7-gene surface marker signature panel that could depict the early surviving MICs of ESCC, including CD44, TACSTD2, TM4SF1, S100A14, RHOD, CST6, and C19orf33. In silico gene ontology (GO) enrichment of the early surviving MICs subpopulation reveals numerous migration and adhesion-related biological processes (p&amp;lt;0.05). In addition to scRNA-Seq, immunohistochemistry (IHC) staining of tumor cells in mice lungs collected in sequential order evidenced the survival heterogeneity of tumor subpopulations. Likewise, flow cytometry fluorescence-activated cell sorting (FACS) of signature-enriched tumor cells was performed to resemble the target MICs. CD44Pos ESCC cells are found to show distinctively greater wound-healing (p&amp;lt;0.01), foci formation (p&amp;lt;0.05), migration (p&amp;lt;0.01) and invasion (p&amp;lt;0.01) ability than CD44Neg cells in vitro, and longer survival patterns (p&amp;lt;0.05) upon implantation in vivo. Multi-color immunofluorescence (mIF) staining of these gene signatures is underway in mice models and clinical samples for comprehensive validation. We anticipate this surface marker signature to accelerate development for clinical diagnosis together with molecular targeting in metastatic ESCC patients. Citation Format: Ching Ngar Wong, Yu Zhang, Beibei Ru, Hong Yu Zhou, Kwong Yiu Lam, Xin Yuan Guan. Single-cell RNA sequencing (scRNA-Seq) from mice model reveals metastatic and stemness signatures in esophageal squamous cell carcinoma (ESCC) [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 5982.

Abstract 960: HAPLN1 increases peritoneal carcinomatosis by inducing tumor cell hyperplasticity

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) frequently metastasizes into the peritoneum forming peritoneal carcinomatosis, which are so far not treatable effectively. Metastasis-initiating cells need to acquire beneficial traits including cell plasticity, immune evasion, dormancy state control and organ colonization. These characteristics can be summarized in broad terms into two main processes, epithelial-to-mesenchymal transition (EMT) and stemness. Hyaluronic acid (HA), an extracellular matrix component, is a crucial factor in regulating these processes in PDAC, but it is so far not successfully targetable. Analyzing publicly available databases by gene set enrichment analysis (GSEA), a signature related to HA binding was enriched in tumor samples compared to normal tissue. Hyaluronan And Proteoglycan Link Protein 1 (HAPLN1) was the top contributor to the enrichment score, being the 8th most enriched gene overall. We found that higher HAPLN1 expression correlated with shorter overall survival and that HAPLN1high patients had both, basal subtype and EMT signatures enriched. Moreover, these patients had a signature for peritoneal metastasis significantly enriched, suggesting a higher risk for peritoneal carcinomatosis. To study the role of HAPLN1 on PDAC in vitro, we stably overexpressed HAPLN1 in the murine PDAC cell line KPC. KPC-HAPLN1 cells expressed more EMT markers, more stem-related genes and changed the proteoglycan production from Aggrecan to Versican, which is known to be pro-metastatic. We found that spheroid formation, a feature of stemness, was improved in KPC-HAPLN1 vs KPC. Additionally, embedding these spheroids into matrigel led to an increased invasion of KPC-HAPLN1 cells. KPC-HAPLN1 cells improved KPC cell invasion capacities when co-cultured, indicating a paracrine effect. In vivo, intraperitoneal injection of luciferase expressing KPC cells resulted in higher luciferase activity when tumor cells expressed HAPLN1. Analyzing the peritoneal lavage (PL) from these mice, we obtained significantly more tumor cells in KPC-HAPLN1 injected mice. RNAseq data of tumor cells isolated from tumor nodules and PL showed that KPC-HAPLN1 cells acquired an increased metastatic potential and a strong immunomodulatory phenotype. Thus, we evaluated the immune cell composition of the PL by flow cytometry. Neutrophil and monocyte percentages were drastically reduced in KPC-HAPLN1 bearing mice. On the contrary, these mice had a significant increase in macrophages, which showed a reduction in pro-inflammatory gene expression. We conclude that HAPLN1 expression in tumor cells promotes a hyperplastic phenotype that facilitates invasion and colonization of the peritoneum, among others by creation of a pro-tumoral immune microenvironment. Citation Format: Lena Wiedmann, Francesca De Angelis Rigotti, Nuria Vaquero-Siguero, Elisa Donato, Elisa Espinet, Andreas Trumpp, Andreas Fischer, Juan Rodriguez-Vita. HAPLN1 increases peritoneal carcinomatosis by inducing tumor cell hyperplasticity [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 960.

12P Any size of lymph node metastasis should be considered N1 in patients with cervical cancer

12P Any size of lymph node metastasis should be considered N1 in patients with cervical cancer