Metastatic Clones Research Articles

Intraperitoneal transfer of microRNA-29b-containing small extracellular vesicles can suppress peritoneal metastases of gastric cancer.

Small extracellular vesicles (sEV) contain various microRNAs (miRNAs) and play crucial roles in the tumor metastatic process. Although miR-29b levels in peritoneal exosomes were markedly reduced in patients with peritoneal metastases (PM), their role has not been fully clarified. In this study, we asked whether the replacement of miR-29b can affect the development of PM in a murine model. UE6E7T-12, human bone marrow-derived mesenchymal stem cells (BMSCs), were transfected with miR-29b-integrating recombinant lentiviral vector and sEV were isolated from culture supernatants using ultracentrifugation. The sEV contained markedly increased amounts of miR-29b compared with negative controls. Treatment with transforming growth factor-β1 decreased the expression of E-cadherin and calretinin with increased expression of vimentin and fibronectin on human omental tissue-derived mesothelial cells (HPMCs). However, the effects were totally abrogated by adding miR-29b-rich sEV. The sEV inhibited proliferation and migration of HPMCs by 15% (p < 0.005, n = 6) and 70% (p < 0.005, n = 6), respectively, and inhibited adhesion of NUGC-4 and MKN45 to HPMCs by 90% (p < 0.0001, n = 5) and 77% (p < 0.0001, n = 5), respectively. MicroRNA-29b-rich murine sEV were similarly obtained using mouse BMSCs and examined for in vivo effects with a syngeneic murine model using YTN16P, a highly metastatic clone of gastric cancer cell. Intraperitoneal (IP) transfer of the sEV every 3 days markedly reduced the number of PM from YTN16P in the mesentery (p < 0.05, n = 6) and the omentum (p < 0.05, n = 6). Bone marrow mesenchymal stem cell-derived sEV are a useful carrier for IP administration of miR-29b, which can suppress the development of PM of gastric cancer.

Abstract P5-13-01: TUMOR PROGRESSION MODEL IN BREAST CANCER WITH BRAIN METASTASIS

Abstract INTRODUCTION: Breast cancer are the main cause of related deaths cancer among women, corresponding to 25% of new cases each year. When diagnosed at early stages, it has an overall five-year survival rate of up to 90%. However, in more advanced stages, survival is reduced to about 24%, with 90% of women in stage IV dying as a result of complications related to metastases. Considering that brain metastasis is an unfavorable prognostic site, and the identification of genetic-molecular profiles in primary tumors and in metastatic sites are a subject poorly described in the literature, we understand that the identification of mutational profiles may contribute to elucidate the genetic-molecular mechanisms associated with tumor progression. AIM: The aim of this study was to identify clonal and subclonal driver mutations that lead to evolution of metastatic clones from a breast cancer progression model. MATERIAL AND METHODS: For tumor progression model, automated extraction of DNA from buffy coat and paraffin samples of breast tumors and paired brain metastases (n=9) was performed. In the present work, we used a subclonal reconstruction model based on the combination of machine learning and population genetics concepts. This proposal is based on the frequency spectrum of each somatic mutation (SNVs or indels), considering VAF (Variant Allele Frequency - ratio of mapped reads of the mutant allele) in relation to the coverage of variant locus, as known as CCF (Cancer Cell Fraction). CCF is defined as the proportion of neoplastic cells that have a certain set of mutations and then is normalized considering the sample purity and the segments with changes in number of DNA copies (Copy Number Alterations). Then, a statistical model based on finite Dirichlet mixtures with mixed distributions is applied. In this model, Beta components capture clonal expansions and population genetics concepts were applied to mutant alleles in each population considering principles of cancer evolution. Finally, confidence was computed using both parametric and non-parametric bootstraps. The functions for building the model are implemented at https://caravagnalab.github.io/mobster/, and for visualization and construction of graphs, packages in R statistical-mathematical environment were used, such as ggplot2, sads, cli, clisymbols, cowplot, crayon, ctree, dndscv, dplyr, magrittr, reshape2, and tidyr. RESULTS: With this model was possible to identify the distribution of clones according to somatic alterations in patients with breast cancer and brain metastasis. It was possible to observe common patterns, such as alterations of the SF3B1 gene as a common ancestral clone in both conditions and the frequency of the AKT1 gene in a subclonal condition. Other genes relevant to breast cancer carcinogenesis, such as PIK3CA and TP53, are found in a different clonal hierarchical pattern between the two conditions. CONCLUSION: This data suggests a model of clonal evolution capable to identify which drivers clones and subclones are involved in the metastatic process. Citation Format: Muriele B. Varuzza, Adriane F. Evangelista, Cristiano P. Souza, Márcia C. Marques. TUMOR PROGRESSION MODEL IN BREAST CANCER WITH BRAIN METASTASIS [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-13-01.

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.

2MO Tracking immunoediting from early to late-stage lung cancer

2MO Tracking immunoediting from early to late-stage lung cancer

SCD5-dependent inhibition of SPARC secretion hampers metastatic spreading and favors host immunity in a TNBC murine model.

Dysregulated fatty acid metabolism interacts with oncogenic signals, thereby worsening tumor aggressiveness. The stearoyl-CoA desaturating enzymes, SCD1 and SCD5, convert of saturated fatty acids to monounsaturated fatty acids. While SCD1 is frequently overexpressed in tumor cells and has been widely studied, SCD5 has both limited expression and poor characterization. Here we evaluated, in vitro and in vivo, the effects of SCD5 overexpression in a metastatic clone of 4T1. The results showed SCD5-driven reprogramming of fatty acid metabolism, involving desaturation of stearic acid to oleic acid, which eventually blocked SPARC secretion. The latter event reduced the aggressiveness of the 4T1 subclone by decreasing the ECM deposition and reverting the Epithelial to Mesenchymal Transition (EMT) status. Variation of the fatty acid profile by SCD5-gene transduction or the direct administration oleic acid reduces the immune suppressive activity of myeloid cells and promoting granulocytic myeloid-derived suppressor cell maturation, eventually favoring T-cell activation. The less immunosuppressive microenvironment generated by SCD5 overexpression was enhanced in Sparc-KO mice, indicating that both extracellular and endogenous SPARC additively regulate myeloid cell-suppressive activities. Overall, our data sheds light on exploring the oleic acid-dependent inhibition of SPARC secretion as a possible mechanism to reduce breast cancer malignancy.

Abstract 6269: A cell double barcoding system for quantitative evaluation of primary tumors &amp; metastasis in animals: Uncovers clonal-specific anti-cancer drug effects

Abstract Imaging in monitoring metastasis in mouse models have low sensitivity and is not quantitative. Cell DNA barcoding, demonstrating high sensitivity and resolution, allows monitoring effects of drugs on the number of tumor and metastatic clones. However, this technology is not suitable for comparison of sizes of metastatic clones in different animals, e.g. drug treated and untreated, due to high biological and technical variability upon tumor and metastatic growth and isolation of barcodes from tissue DNA. Yet, both numbers of clones and their sizes are critical parameters for analysis of drug effects. Here we developed a modification of the barcoding approach for monitoring drug effects on tumors and metastasis that is quantitative, highly sensitive and highly reproducible. This novel cell double barcoding system allows simultaneously following the fate of two or more cell variants or cell lines in xenograph models in vivo, and also following the fates of individual clones within each of these populations. This system allows comparing effects of drugs on different cell populations, and thus normalizing drug effects by drug-resistant lines, which corrects for both biological and technical variabilities and significantly increases the reproducibility of results. Using this barcoding system, we uncovered that effects of a novel DYRK1B kinase inhibitor FX9847 on primary tumors and metastasis is clone-dependent, while a distinct drug osimertinib demonstrated clone-independent effects on cancer cell populations. Overall cell double barcoding approach can significantly enrich our understanding of drug effects in basic research and preclinical studies. Citation Format: Arkadi Hesin, Santosh Kumar, Valid Gahramanov, Maria Becker, Maria Vilenchik, Ilya Alexandrov, Julia Yaglom, Michael Sherman. A cell double barcoding system for quantitative evaluation of primary tumors &amp; metastasis in animals: Uncovers clonal-specific anti-cancer drug effects [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 6269.

Abstract 3792: TRACERx: Mapping the evolution of metastases in non-small cell lung cancer

Abstract Background: Primary lung cancer is the leading cause of cancer-related mortality, with metastatic disease being responsible for the majority of deaths. To gain insight into the lethal process of metastasis, we report on the longitudinal evolutionary analysis of the TRACERx 421 paired primary-metastasis cohort. Methods: 712 tumor samples, of which 485 were primary tumor and 227 were metastatic samples, from 129 metastatic non-small cell lung cancer (NSCLC) patients with detailed clinical annotation were collected from 18 UK hospital sites and whole exome sequenced. Mutations and copy number events were integrated to resolve the evolutionary history of each tumor. Results: We observe that metastases generally diverge relatively late in molecular time, after the majority of mutations in the primary tumor have accumulated, with a substantial minority (33%) diverging prior to the last clonal sweep in the primary tumor. For this minority of cases, divergence is estimated to have occurred at tumor volumes below the limit of computed tomography (CT) detection. Our extensively sampled cohort reveals that sampling bias may result in erroneous inference of metastatic trajectories. 79% of metastases diverging after the last clonal sweep in the primary tumor would be misclassified as diverging prior to the last clonal sweep if only a single region of the primary tumor is considered. Patterns of dissemination range from monoclonal, involving a single metastatic clone (68% - where metastatic potential is likely acquired once in the life-history of the tumor), to polyclonal and polyphyletic (17%), where metastatic potential may have arisen multiple times during lung cancer development, or at a single time point early in the development of the tumor. We find that thoracic lymph node disease resected at surgery was responsible for less than 20% of subsequent disease dissemination, suggesting that lymph nodes likely represent a hallmark of metastatic potential rather than a gateway to further metastases. Furthermore, we observe that clones which seed the metastases are generally dominant within the primary, reflecting positive selection and acquisition of subclonal mutations in specific cancer genes (e.g. RB1, PIK3CA). In squamous cell carcinomas, we find that non-metastatic primary tumors show no significant evidence of positive subclonal selection. We find that 35% of metastases harbor driver mutations not identified in the primary tumor and identify somatic copy number alterations that are enriched in metastases (e.g. CCND1 gains in lung adenocarcinoma). Conclusions: These data highlight the potential to apply evolutionary measures to primary tumors to predict metastatic risk, the limitations to current screening approaches particularly for early tumor divergence, and the importance of future precision adjuvant therapies to target disseminated micro-metastatic clones. Citation Format: Ariana Huebner, Maise Al Bakir, Carlos Martinez Ruiz, Kristiana Grigoriadis, Thomas B. Watkins, Oriol Pich, David A. Moore, Selvaraju Veeriah, Sophia Ward, Joanne Laycock, Diana Johnson, Andrew Rowan, Maryam Razaq, Mita Akther, Cristina Naceur-Lombardelli, Sonya Hessey, Michelle Dietzen, Emma Colliver, Alexander M. Frankell, Emilia Lim, Takahiro Karasaki, Christopher Abbosh, Crispin T. Hiley, Mark S. Hill, Daniel Cook, Gareth Wilson, TRACERx consortium, Allan Hackshaw, Nicolai J. Birkbak, Simone Zaccaria, Mariam Jamal-Hanjani, Charles Swanton, Nicholas McGranahan. TRACERx: Mapping the evolution of metastases in non-small cell lung cancer [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 3792.

Association of NR2F1, a tumor dormancy marker, with cancer cell proliferation and lymph node metastasis, and expression in cancer-associated fibroblasts in breast cancer.

e12550 Background: It is now well accepted that some cancer cells remain in quiescent state and survive through adjuvant systemic treatments, also known as tumor dormancy, which preserve the potential for late recurrence. NR2F1 is a known biomarker for dormant cancer cells, which suppress cell proliferation in experimental models. Here, we hypothesized that patient breast cancer (BC) with high NR2F1 gene expression is associated with less cell proliferation and worse survival. Methods: More than 7000 patients from 3 large-scale primary and 5 metastatic BC patient cohorts and 2 single-cell sequencing cohorts were analyzed. Results: NR2F1 expression was upregulated in dormant cell lines and in bone metastatic clone cells compared with mother 4T1 cells. High NR2F1 BC enriched multiple metastasis-related pathways such as TGFβ, EMT, angiogenesis, as well as cancer stem cell-related signaling (NOTCH, HEDGEHOG, and Wntβ-catenin). As expected, all 5 cell proliferation-related gene sets including E2F Targets and G2M Checkpoints were enriched in low NR2F1 BC, and so was MKI67 expression. Unexpectedly, there were no consistent association between NR2F1 expression and survival among the 3 cohorts. Three out of four cohorts demonstrated significantly high NR2F1 expression in primary BC with lymph node metastasis. There was no difference in NR2F1 expression in primary BC by the distant metastasis in the four cohorts, by the metastatic sites, or between the primary and metastatic BC neither. Given the discrepancy between the biology and clinical outcome of high NR2F1 BC, we performed the functional analysis exclusively . Surprisingly, there were no consistent enrichment of metastasis-related gene sets nor downregulation of cell proliferation-related gene sets to high NR2F1 expression cancer cells, unlike the analyses of bulk tumors. Intratumoral heterogeneity, HRD, proliferation score, and mutation load were all less in high NR2F1 BC, along with IFN-γ response and TIL fractions. Infiltration of immune cells such as Th1 and Th2 T-cells, M1 and M2 macrophages were all less, while stromal cells such as adipocytes, fibroblasts, stromal cells and endothelial cells were more abundant in high NR2F1 BC. Surprisingly, NR2F1 was barely expressed in cancer cells and was most highly expressed in cancer-associated fibroblasts consistently regardless of subtypes in two independent single-cell sequence cohorts. The expression of NR2F1 was higher in inflammatory than myofibroblastic CAFs, and that of CAFs, but not cancer cells, was associated with lymph node metastasis or advanced stage. Conclusions: The tumor dormancy marker NR2F1 expression in primary BC is associated with mechanism of metastasis and suppression of cancer growth, but is most expressed in cancer-associated fibroblasts in the tumor microenvironment.

PO-1185 Targeting therapy-resistant metastatic clones with SBRT in oligoprogressive breast cancer

PO-1185 Targeting therapy-resistant metastatic clones with SBRT in oligoprogressive breast cancer

A Cell Double-Barcoding System for Quantitative Evaluation of Primary Tumors and Metastasis in Animals That Uncovers Clonal-Specific Anti-Cancer Drug Effects.

Simple SummaryThe main problem in treating advanced cancers is a metastatic spread when individual cancer cells leave the primary tumor and colonize to distant organs. In drug development, it is important to quantitatively assess effects of novel drug candidates on both primary tumors and metastasis. Unfortunately, current methods of monitoring metastasis in mouse models have low sensitivity and are not quantitative. Here, we developed a methodology to monitor drug effects on metastasis that is quantitative and has a very high sensitivity and resolution. In fact, it allows monitoring effects of drugs on individual cancer cells in animals.Imaging in monitoring metastasis in mouse models has low sensitivity and is not quantitative. Cell DNA barcoding, demonstrating high sensitivity and resolution, allows monitoring effects of drugs on the number of tumor and metastatic clones. However, this technology is not suitable for comparison of sizes of metastatic clones in different animals, for example, drug treated and untreated, due to high biological and technical variability upon tumor and metastatic growth and isolation of barcodes from tissue DNA. However, both numbers of clones and their sizes are critical parameters for analysis of drug effects. Here we developed a modification of the barcoding approach for monitoring drug effects on tumors and metastasis that is quantitative, highly sensitive and highly reproducible. This novel cell double-barcoding system allows simultaneously following the fate of two or more cell variants or cell lines in xenograft models in vivo, and also following the fates of individual clones within each of these populations. This system allows comparing effects of drugs on different cell populations and thus normalizing drug effects by drug-resistant lines, which corrects for both biological and technical variabilities and significantly increases the reproducibility of results. Using this barcoding system, we uncovered that effects of a novel DYRK1B kinase inhibitor FX9847 on primary tumors and metastasis is clone-dependent, while a distinct drug osimertinib demonstrated clone-independent effects on cancer cell populations. Overall, a cell double-barcoding approach can significantly enrich our understanding of drug effects in basic research and preclinical studies.

Spatial and temporal intratumour heterogeneity has potential consequences for single biopsy-based neuroblastoma treatment decisions

Intratumour heterogeneity is a major cause of treatment failure in cancer. We present in-depth analyses combining transcriptomic and genomic profiling with ultra-deep targeted sequencing of multiregional biopsies in 10 patients with neuroblastoma, a devastating childhood tumour. We observe high spatial and temporal heterogeneity in somatic mutations and somatic copy-number alterations which are reflected on the transcriptomic level. Mutations in some druggable target genes including ALK and FGFR1 are heterogeneous at diagnosis and/or relapse, raising the issue whether current target prioritization and molecular risk stratification procedures in single biopsies are sufficiently reliable for therapy decisions. The genetic heterogeneity in gene mutations and chromosome aberrations observed in deep analyses from patient courses suggest clonal evolution before treatment and under treatment pressure, and support early emergence of metastatic clones and ongoing chromosomal instability during disease evolution. We report continuous clonal evolution on mutational and copy number levels in neuroblastoma, and detail its implications for therapy selection, risk stratification and therapy resistance.

TAMI-04. OLFACTORY RECEPTOR 5B21 DRIVES BREAST CANCER BRAIN METASTASIS THROUGH STAT3/NFΚB/CEBPΒ PATHWAY

Abstract Olfactory receptors (ORs), responsible for the sense of smell, play an essential role in various physiological processes outside the nasal epithelium, including cancer. In breast cancer, however, the expression and function of ORs remain understudied. We established a breast cancer metastasis model by intracardiac injection of MDA-MB-231 (231P) in immunocompromised mice and produced a series of derivative cell lines from developed metastatic sites, including the brain-seeking clone (231Br). We examined the significance of ORs transcript abundance in primary and metastatic breast cancer to different tissues, including the brain, bone, and lung. While 20 OR transcripts were differentially expressed in distant metastases, OR5B21 displayed high expression in all three metastatic sites with respect to the primary tumor, especially in brain metastasis with 13 fold higher than the primary site. Metastatic clones showed distinguishing higher invasion biological characteristics compared to parental cells in vivo and in vitro. Knockdown of OR5B21 significantly decreased the invasion and migration of MDA-MB-231 Brain-seeking metastatic cell as well as metastasis to different organs, including the brain, while overexpression of OR5B21 had the opposite effect. Mechanistically, OR5B21 expression was associated with epithelial to mesenchymal transition through the STAT3/NFkB/CEBPβ signaling pathway. We propose OR5B21 (and potentially other ORs) as a novel oncogene contributing to breast cancer brain metastasis and a potential target for adjuvant therapy.

Androgen receptor (AR) heterogeneity in prostate cancer and therapy resistance.

Androgen receptor (AR), a ligand-dependent nuclear transcription factor and a member of steroid hormone receptor family, plays an important role in prostate organogenesis by regulating epithelial differentiation and restricting cell proliferation. Although rarely mutated or amplified in treatment-naïve prostate cancer (PCa), AR signaling drives tumor growth and as a result, therapies that aim to inhibit AR signaling, called ARSIs (AR signaling inhibitors), have been in clinical use for >70 years. Unfortunately, the clinical efficacy of ARSIs is short-lived and the majority of treated patients develop castration-resistant PCa (CRPC). Numerous molecular mechanisms have been proposed for castration resistance; however, the cellular basis for CRPC emergence has remained obscure. One under-appreciated cellular mechanism for CRPC development is the AR heterogeneity that pre-exists in treatment-naive primary tumors, i.e., although most PCa cells express AR (i.e., AR+), there is always a population of PCa cells that express no/low AR (i.e., AR-/lo). Importantly, this AR heterogeneity becomes accentuated during ARSI treatment and highly prominent in established CRPC. Here, we provide a succinct summary of AR heterogeneity across the PCa continuum and discuss its impact on PCa response to treatments. While AR+ PCa cells/clones exhibit exquisite sensitivities to ARSIs, AR-/lo PCa cells/clones, which are greatly enriched in stem cell signaling pathways, display de novo resistance to ARSIs. Finally, we offer several potential combinatorial strategies, e.g., ARSIs with stem cell targeting therapeutics, to co-target both AR+ and AR-/lo PCa cells and metastatic clones.

Single-cell exome sequencing reveals multiple subclones in metastatic colorectal carcinoma

BackgroundColorectal cancer (CRC) is a major cancer type whose mechanism of metastasis remains elusive.MethodsIn this study, we characterised the evolutionary pattern of metastatic CRC (mCRC) by analysing bulk and single-cell exome sequencing data of primary and metastatic tumours from 7 CRC patients with liver metastases. Here, 7 CRC patients were analysed by bulk whole-exome sequencing (WES); 4 of these were also analysed using single-cell sequencing.ResultsDespite low genomic divergence between paired primary and metastatic cancers in the bulk data, single-cell WES (scWES) data revealed rare mutations and defined two separate cell populations, indicative of the diverse evolutionary trajectories between primary and metastatic tumour cells. We further identified 24 metastatic cell-specific-mutated genes and validated their functions in cell migration capacity.ConclusionsIn summary, scWES revealed rare mutations that failed to be detected by bulk WES. These rare mutations better define the distinct genomic profiles of primary and metastatic tumour cell clones.

Abstract 3127: A transcriptional lineage tracing approach to unravel crucial gene expression patterns in breast cancer progression

Abstract Breast cancer represents a major clinical hurdle, with intra-tumor heterogeneity and metastatic outcome accounting for poor prognosis. Traditionally, its poly-clonal structure was investigated through the genetic barcoding of cells followed by the tracing of the offspring. However, this analysis does not allow a solid investigation of the transcriptional profile associated to clones throughout tumor progression. In this work, we exploited a recently published library of expressed barcodes to perform both clonal and transcriptional tracing in breast cancer by single-cell RNA sequencing. To this aim, human breast cancer cell line MDA-MB-231 was transduced with the above mentioned library and orthotopically transplanted in immune-deficient mice. To analyze our data we generated a pipeline aimed at identifying the barcodes at single-cell level and subsequently sequenced more than 20000 cells in six independent primary tumors. Our clonal distribution analysis strongly suggested that clones within the same tumor possess different growth potential. Furthermore, we found that breast tumors show a transcriptional heterogeneity characterized by two major transcriptional patterns: highly proliferating cells on one side, and stressed and dormant cells on the other. Notably, breast cancer clones were shown to belong to one of these two patterns, therefore suggesting that primary tumors are composed of both proliferative and non-proliferative clones. Interestingly, although the non-proliferative clones outnumbered the proliferative ones, the two different transcriptional patterns do not differ in terms of absolute number of cells. Analyses of breast cancer metastases to lungs and liver revealed that the clones that spread metastases belong to both the transcriptional patterns identified in the primary tumor. Interestingly, the metastatic clones were found to be rare in the primary tumors and shared between the two colonized organs. In addition, metastases recapitulated the same transcriptional heterogeneity of primary tumors, despite being composed of a much lower number of clones. Altogether, these findings represent an intriguing launching pad for the identification of the transcriptional markers whose inhibition may counter the metastatic outcome. Citation Format: Niccolò Roda, Roman Hillje, Stefano Cheloni, Valentina Gambino, Giada Blandano, Paolo Falvo, Francesco Bertolini, Pier Giuseppe Pelicci. A transcriptional lineage tracing approach to unravel crucial gene expression patterns in breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3127.

Abstract 2174: The genomic and anti-tumor immune evolution of breast cancer

Abstract Background: Immunotherapy has changed the paradigm for a fraction of cancer patients. However, the evolution of tumor and immune microenvironment may uncover therapy resistance and immune evasion and its impact on clinical outcome. Here, in order to analyze the genomic and anti-tumor immune evolution, we performed extensive characterization of a dataset of a triple negative breast cancer patient including longitudinal and synchronous metastases (mets) and blood samples, collected over 2,039 days (d) of clinical follow-up. Methods: Multi-platform profiling included whole exome seq, RNAseq, targeted multiplex gene expression, immunohistochemistry, and ELISpot T-cell assay of longitudinal chest wall biopsies (n=17 samples) and peripheral blood (n=14 samples), synchronous multi-regional mets at research autopsy (n=20 mets) and primary tumor analysis. State-of-the-art bioinformatics was applied to the data. Results: Tumor mutation burden (TMB) estimates from the immunotherapy naïve chest wall lesion (M1) (d799) and 20 multi-regional mets (immunotherapy experienced, IE-mets) (d2,033) were 1.5 mut/mb and had median of 3.9 mut/mb, respectively (delta TMB 1.6 mut/mb). There were 73 neoantigens in M1 (subclonal/clonal ratio=0.4), and a median of 226 neoantigens per IE-mets (subclonal/clonal ratio=1.1). TP53 T256fs was validated as a T-cell specific clonal neoantigen and was targeted as liquid biopsy cell-free tumor DNA since cancer diagnosis. Clonal ordering and clonal evolution models for M1 and IE-mets evidenced that clustered mutations shared the same evolutionary history but with a mixed pattern of seeding: metastatic clones were shed by polyclonal and monoclonal seeding. Distinct seeding events occurred from early M1 to late IE-mets, and among late IE-mets (CI 0.95, p&amp;lt; 0.0001). Longitudinal tumor inflammation (IFNy signaling) and antigen presenting machinery (APM) signatures defined immunophenotype statuses (i-inflamed, i-desert, i-excluded). They were modulated over clinical responses towards immune evasion and higher tumor cell proliferation. Immunophenotype statuses were also assigned to IE-mets that showed an overall heterogeneity and significant positive correlation to TMB (p=0.012), but not with the number of predicted neoantigens. M1 had HLA machinery status intact at the genomic level. IE-mets immunophenotyped as i-desert displayed statistically significant HLALOH (p&amp;lt;0.05), indicating APM failure. However, an i-inflamed monoclonal IE-met evolving from M1 kept HLA status intact. This suggested APM machinery failure in IE-mets likely went through clonal evolution as a result of an immunoediting process. Conclusion: By analysis of metastases genomes, we show that the immune tumor microenvironment shapes triple negative breast cancer progression by promoting and activating immune evasion mechanisms, culminating in heterogeneous lethal cancer. Citation Format: Juan Blanco-Heredia, Carla Anjos Souza, Samuel Gonçalves-Ribeiro, Maria Gonzalez-Cao, Maurizio Callari, Francesc Tresserra, Rafael Rossell, Carlos Caldas, Leticia De Mattos-Arruda. The genomic and anti-tumor immune evolution of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2174.

Abstract SY02-02: Understanding evolution of metastatic disease in renal cancer and melanoma

Abstract While our knowledge of the evolutionary features of primary tumors has grown exponentially over the last few years our understanding of evolution of metastases is more limited and many open questions remain, regarding the timing of metastatic dissemination, the acquisition of metastatic competence and the underpinning of the variety of metastatic phenotypes observed in the clinic from latent metastases to solitary and oligo-metastases to widespread metastases. We have leveraged unique translational protocols that allow interrogation of tumor evolution from the earliest stages of diseases to death to address some of these questions. In the context of renal cell cancers we show that the mode of evolution at the primary tumor site determines the tempo and distribution of systemic metastases; that the features that distinguish metastasis-competent clones included genome instability and aneuploidy; and that metastatic competence often emerges in the centre of the primary tumor. In the context of melanoma we observe a wide spectrum of metastatic seeding that includes organ-specific metastatic clones, monophyletic and polyphyletic seeding and polyclonal mixing at metastatic sites. We observe progressive increase in aneuploidy and occurrence of whole genome doubling as melanoma metastases progress and resist treatment suggesting this as a a mechanism of immune-evasion and treatment resistance. Citation Format: Samra Turajlic. Understanding evolution of metastatic disease in renal cancer and melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr SY02-02.

Abstract LB226: Maternal microbiome protects host against clonal de novo transformation, early onset systemic metastasis, and sudden death

Abstract The role of maternal microbiome transmitted at birth in cancer control is poorly understood. We have developed the first germfree B6 mouse model of breast cancer to investigate the role of the maternal microbiome in controlling oncogenic/metastatic frequencies of pro-oncogenic mammary cells. In this model, a DNA barcoded, primitive normal mouse mammary epithelial cell encoding MMTV-PyMT oncogene, was transplanted in large numbers into conventional or germfree B6 mice. Next-gen sequencing analysis of the DNA barcodes in tissues enabled us to clonally track millions of cells and measure the frequency and growth dynamics of clones at the primary site and their systemic distribution in circulation and all vital organs, generating an unprecedented high-definition map of cancer progression. Our results show that in conventional B6 mice with maternal microbial transmission at-birth, a small fraction (~0.01%) of transplanted cells transform de novo and produce slow growing, late-onset benign tumors (mouse median survival of &amp;gt; 1 year). In contrast, in germfree B6 mice, a &amp;gt;10-fold higher frequency of cells transform de novo and generate early-onset, highly aggressive metastatic clones, and are frequently associated with features leading to early euthanasia or endpoint (i.e. sudden death, intracardiac metastasis, paralysis, swollen abdomen, early multiorgan aggressive metastasis) (median survival of ~4 months; p&amp;lt;0.005 vs conventional mice). However, postnatal intervention with healthy fecal implants reconstituted gut microbiota in germfree B6 mice and significantly restored protective effects against cancer progression and sudden death, as well as improved quality/duration of life (median survival of &amp;gt;1 year; p&amp;lt;0.005 vs germfree mice; p=ns vs conventional mice). We found maternal microbial transmission fully protected against systemic metastasis. GC-MS analysis identified significantly distinct primary tumor metabolome associated with conventional (non-metastatic) mice vs germfree (metastatic) mice. Metagenomic analysis of fecal samples from a subset of tumor bearing conventional (non-metastatic) mice vs fecal implanted (late-onset, less aggressive metastatic) germfree mice identified significant association between metastasis and altered microbial communities. These findings using novel germfree mouse model of metastasis and cellular DNA barcoding technology provide first direct evidence that diverse microbial communities transmitted at birth play a critical role in protecting the host from clonal de novo mammary cell transformation and progression to systemic disease and thereby improving quality and duration of life. Citation Format: Nagarajan Kannan, Syed Mohammed Musheer Aalam, Xiaojia Tang, Krishna Kalari, Purna Kashyap, Jun Chen, Stephen Johnson, Anguraj Sadanandam, Mark Sherman. Maternal microbiome protects host against clonal de novo transformation, early onset systemic metastasis, and sudden death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB226.

Nerve growth factor receptor increases the tumor growth and metastatic potential of triple-negative breast cancer cells.

Cellular heterogeneity and the lack of metastatic biomarkers limit the diagnosis of and development of therapies for metastatic triple-negative breast cancer (TNBC). Thus, development of new clinically relevant markers is urgently needed. By using RNA-seq analysis, we found that nerve growth factor receptor (NGFR) was highly expressed in metastatic lung clones of MDA-MB-231 cells. This high level of NGFR expression was necessary for TNBC cells to grow into tumor spheres under nonadhesive conditions, resist anoikis, promote primary tumor growth and increase metastasis in mice. NGFR was also expressed at a high level in a greater number of TNBC patients (45%) than non-TNBC patients (23%), enriched in higher grade tumors, and negatively correlated with the overall survival of TNBC patients. Mechanistic analysis indicated that NGFR exerted its prometastatic effects by binding with neurotrophic receptor tyrosine kinase 3 (TrkC) mainly through a ligand-independent manner, which activated the MEK-ERK1-ZEB1 and PI3K-AKT signaling pathways, increased the level of fibronectin, and decreased the expression of PUMA. Notably, we observed that NGFR expression in TrkC-positive metastatic clones reduced cellular sensitivity to anti-Trk therapy. Moreover, WNT family member 5a (WNT5A) and TrkC activated NGFR transcription in a ZEB1-dependent manner. Taken together, this study identified NGFR as a novel driver for transforming TNBC into higher grade metastatic tumors. Our findings provide the basis for the future development of NGFR as a diagnostic and prognostic marker for determining the metastatic potential of TNBC and as a therapeutic target for treating TNBC patients.

PathFinder: Bayesian inference of clone migration histories in cancer.

Metastases cause a vast majority of cancer morbidity andmortality. Metastatic clones are formed by dispersal of cancer cells to secondary tissues, and are not medically detected or visible until later stages of cancer development. Clone phylogenies within patients provide a means of tracing the otherwise inaccessible dynamic history of migrations of cancer cells. Here, we present a new Bayesian approach, PathFinder, for reconstructing the routes of cancer cell migrations. PathFinder uses the clone phylogeny, the number of mutational differences among clones, and the information on the presence and absence of observed clones in primary and metastatic tumors. By analyzing simulated datasets, we found that PathFinder performes well in reconstructing clone migrations from the primary tumor to new metastases as well as between metastases. It was more challenging to trace migrations from metastases back to primary tumors. We found that a vast majority of errors can be corrected by sampling more clones per tumor, and by increasing the number of genetic variants assayed per clone. We also identified situations in which phylogenetic approaches alone are not sufficient to reconstruct migration routes.In conclusion, we anticipate that the use of PathFinder will enable a more reliable inference of migration histories and their posterior probabilities, which is required to assess the relative preponderance of seeding of new metastasis by clones from primary tumors and/or existing metastases. PathFinder is available on the web at https://github.com/SayakaMiura/PathFinder.