Dormant Disseminated Tumor Cells Research Articles

Advances of Fundamental Research on Traditional Chinese Medicine in Regulation of Tumor-associated Macrophages for the Prevention and Treatment of Lung Cancer Metastasis

Lung cancer is the leading cause of cancer-related deaths worldwide, with metastasis being the primary cause of mortality in lung cancer patients, and its prevention and control efficacy remain limited. In recent years, immunotherapy has emerged as a promising direction for overcoming the bottleneck of metastasis. Macrophages, as essential components of innate immunity, participate in the entire process of tumor initiation and progression. Tumor-associated macrophages (TAMs) represent the most abundant immune population in the tumor microenvironment (TME), displaying both anti-tumor M1-like and pro-tumor M2-like phenotypes. The latter promotes tumor invasion and metastasis, angiogenesis, lymphangiogenesis, immune suppression, and reactivation of dormant disseminated tumor cells (DTCs), thereby facilitating tumor metastasis. In recent years, traditional Chinese medicine (TCM) has shown significant efficacy in inhibiting tumor metastasis and has been extensively validated. It exerts anti-tumor effects by reducing the recruitment of TAMs, inhibiting M2-like polarization, and modulating cytokines and proteins in the TME. This paper reviews the relationship between TAMs and lung cancer metastasis, elucidates the targets and mechanisms of TCM in regulating TAMs to prevent and treat lung cancer metastasis, aiming to provide insights into lung cancer prevention and treatment. .

Abstract A074: Targeting dormant disseminated tumor cells and their permissive niche by pro-resolving mediators derived from resolution-phase macrophages

Abstract Metastatic breast cancer can recur years after primary tumor resection and adjuvant therapy and appears to arise from quiescent disseminated tumor cells (QDTC) that persist at distant sites and resist conventional therapies. To date there are no treatments to target QDTCs. Previously, the fibrotic-like microenvironment enriched with Type I collagen (Col-I) was shown to be required for the switch of QDTC to metastatic outgrowth in lungs of mice. Here, we examined whether soluble mediators secreted by ex-vivo generated pro-resolving CD11blow macrophages (CM-Mres) will reinstate resolution of inflammation thus inhibiting the establishment of the fibrotic-like-niche and in turn prevent the emergence of QDTC to metastatic outgrowth. Our findings indicate that CM-Mres promoted immune silencing of alveolar macrophages at the metastatic site where dormant DTCs reside. Immune silencing is one of the hallmarks of resolution of inflammation. Furthermore, CM-Mres inhibited the establishment of a fibrotic-like niche resulting in the inhibition of the metastatic outgrowth in vitro and in vivo. This was due to inhibition of fibroblasts differentiation to myofibroblasts independent of TGFbeta1 canonical signaling and abolishment of Col-I expression. Furthermore, CM-Mres deactivated myofibroblasts as part of the resolution process by inducing an increase in reactive oxygen species (ROS) via activation of NADPH oxidase. This in turn induced DNA damage leading to Go/G1 arrest and intrinsic apoptosis. Similarly, apoptosis mediated by oxidative stress was induced by CM-Mres in dormant and outbreaking QDTCs. Taken together our results demonstrate for the first time that by reinstating resolution of inflammation via physiological mediators, secreted by pro-resolving macrophages, can prevent metastatic outgrowth of QDTCs at their permissive site. Since conventional therapies fail to eradicate dormant DTCs and their transition to clinical metastasis, our findings hold great promise in the quest to identify novel strategies to prevent or treat recurrent metastatic disease. Citation Format: Odelya Gilon, Keren Weidenfeld, Hadell Samara, Yonatan Feuermann, Sagie Schif-Zuck, Sergei Butenko, Edmond Sabo, Amiram Ariel, Dalit Barkan. Targeting dormant disseminated tumor cells and their permissive niche by pro-resolving mediators derived from resolution-phase macrophages [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A074.

A Live Tracker of Dormant Disseminated Tumor Cells.

The high prevalence of dormant disseminated tumor cells (DTCs) persisting systemically in patients with metastatic cancer is a major threat to long-lasting cure (Aguirre-Ghiso, Nat Rev Cancer 7:834-846, 2007; Klein, Nat Rev Cancer 20(11):681-694, 2020; Lyden et al. Cancer Cell 40:787-791, 2022). Despite its clinical significance, the study of what drives DTCs in and out of dormancy while they linger in distant sites has been challenged by the lack of tools to find and follow dormant DTCs inside a living organism. Here, leveraging the fact that dormant DTCs are mostly quiescent, we describe a live cell reporter to distinguish dormant from cycling DTCs (Correia, Nat Rev Cancer 22(7):379, 2022; Correia et al. Nature 594(7864):566-571, 2021). Cancer cell lines are engineered to coexpress a luciferase-tdTomato reporter and a fluorescent fusion protein of mVenus with a mutant form of the cell cycle inhibitor p27 (mVenus-p27K-) that identifies quiescent cells. When implanted in animal models or assembled in cocultures in vitro, labeled cells can be imaged longitudinally over time or retrieved alive alongside their surrounding microenvironment for downstream gene, protein, and metabolite profiling, allowing the mapping of tissue-specific determinants of cancer dormancy and metastasis.

13C Tracer Analysis and Metabolomics in Dormant Cancer Cells.

Over the last two decades, major advances in the field of tumor dormancy have been made. Yet, it is not completely understood how dormant disseminated tumor cells survive and transition to a proliferative state to generate a metastatic lesion. On the other hand, metabolic rewiring has been shown to influence metastasis development through the modulation of both intracellular signaling and the crosstalk between metastatic cells and their microenvironment. Thus, studying the metabolic features of dormant disseminated tumor cells has gained importance in understanding the dormancy process. Here, we describe a method to perform metabolomics and 13C tracer analysis in 3D cultures of dormant breast cancer cells.

Immune evasion of dormant disseminated tumor cells is due to their scarcity and can be overcome by T cell immunotherapies

The period between "successful" treatment of localized breast cancer and the onset of distant metastasis can last many years, representing an unexploited window to eradicate disseminated disease and prevent metastases. We find that the source of recurrence-disseminated tumor cells (DTCs) -evade endogenous immunity directed against tumor neoantigens. Although DTCs downregulate major histocompatibility complex I, this does not preclude recognition by conventional Tcells. Instead, the scarcity of interactions between two relatively rare populations-DTCs and endogenous antigen-specific Tcells-underlies DTC persistence. This scarcity is overcome by any one of three immunotherapies that increase the number of tumor-specific Tcells: Tcell-based vaccination, or adoptive transfer of Tcell receptor or chimeric antigen receptor Tcells. Each approach achieves robust DTC elimination, motivating discovery of MHC-restricted and -unrestricted DTC antigens that can be targeted with Tcell-based immunotherapies to eliminate the reservoir of metastasis-initiating cells in patients.

Abstract B038: Prostate cancer (PCa) cell dormancy in bone depends on physical contacts between PCa cells and osteoblasts and can be induced via FAK inhibition

Abstract Advanced prostate cancer (PCa) in patients still progresses to metastatic relapse after curative treatment such as primary tumor removal. This is likely due to the early disseminated PCa cells that stayed dormant initially and got reactivated to proliferate into overt metastases. However, we don’t know when and to which organs PCa cells disseminate, whether and how primary tumor removal affects the dissemination and dormancy. To answer these, we injected C4-2B cells subcutaneously into immunodeficient NSG mice and randomized the mice for either having the tumors removed at week 2 or keeping the tumors till the endpoints. We then euthanized the mice and semi-quantified the disseminated C4-2B cells in various mouse organs via human-specific genomic PCR at weeks 2/6/8. We serendipitously found PCa metastatic recurrence at a distant site in one mouse at 5 weeks post removal of the C4-2B PCa subcutaneous tumor, suggesting that the mice without observed recurrence may have dormant disseminated tumor cells (DTC). We further observed that tumor removal limited DTC in the bones, specifically, in the bone cortex. In clinic, metastatic PCa tumors were found in patient bone marrows and bone marrow DTC presence was associated with poor prognosis. Therefore, we hypothesized that cells in the bone cortex induce and maintain DTC dormancy. We first explored the dormancy-inducing effects of various types of cells in bone. We co-cultured C4-2B cells in direct contact with osteoblasts MC3T3-E1, macrophages Raw264.7, mesenchymal progenitors OP-9, fibroblasts NIH3T3, or endothelial cells HUVEC. The dormancy phenotypes such as proliferation and marker gene changes of the co-cultured C4-2B cells were compared to C4-2B cells cultured alone. We found that only MC3T3-E1 cells, but not other cells, inhibited C4-2B proliferation, increased the expression of dormancy markers such as NR2F1, and decreased the expressions of proliferation markers such as Ki67 and Cyclin D1, suggesting that osteoblasts induced C4-2B dormancy. Direct physical contacts were necessary as dormancy phenotypes were not observed in C4-2B cells treated with the conditioned media of MC3T3-E1 cells cultured alone, nor in C4-2B cells in transwell co-culture with MC3T3-E1 cells. We then performed RNA-Seq in C4-2B/MC3T3-E1 co-culture to profile the dormancy signature, which was loaded into a novel artificial intelligence (AI) platform to screen for drugs mimicking this signature. One candidate from the AI pipeline, PF-562271, a focal adhesion kinase (FAK) inhibitor, was validated with dormancy-mimicking effect for C4-2B cells in vitro. Further studies are focusing on the in vivo effects and the mechanisms of action of PF-562271. Our study profiled the C4-2B PCa cell disseminations post primary tumor removal. We defined a unique osteoblast-induced dormancy signature and revealed physical contact requirement in dormancy. We also recapitulated osteoblast-induced PCa cell dormancy in vitro using an FAK inhibitor, which could potentially inhibit PCa bone metastatic progression and recurrence clinically. Citation Format: Ruihua Liu, Shang Su, Ke Liu, Jing Xing, Mary Stangis, Diego P. Jacho, Eda D. Yildirim-Ayan, Cara M. Gatto-Weis, Haiquan Yu, Bin Chen, Xiaohong Li. Prostate cancer (PCa) cell dormancy in bone depends on physical contacts between PCa cells and osteoblasts and can be induced via FAK inhibition [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B038.

Age-associated microenvironmental changes highlight the role of PDGF-C in ER+ breast cancer metastatic relapse

Patients with estrogen receptor (ER)-positive breast cancer are at risk of metastatic relapse for decades after primary tumor resection and treatment, a consequence of dormant disseminated tumor cells (DTCs) reawakening at secondary sites. Here we use syngeneic ER+ mouse models in which DTCs display a dormant phenotype in young mice but accelerated metastatic outgrowth in an aged or fibrotic microenvironment. In young mice, low-level Pdgfc expression by ER+ DTCs is required for their maintenance in secondary sites but is insufficient to support development of macrometastases. By contrast, the platelet-derived growth factor (PDGF)-Chi environment of aging or fibrotic lungs promotes DTC proliferation and upregulates tumor cell Pdgfc expression stimulating further stromal activation, events that can be blocked by pharmacological inhibition of PDGFRα or with a PDGF-C-blocking antibody. These results highlight the role of the changing microenvironment in regulating DTC outgrowth and the opportunity to target PDGF-C signaling to limit metastatic relapse in ER+ breast cancer.

Abstract SY31-02: Revisiting NK cell immunity to prevent metastasis

Abstract Metastases arising in distant tissues continue to cause the majority of all cancer-related deaths. The uncertainty of whether and when metastases will occur poses a major challenge to treatment effectiveness. In many cancer patients, persistent disseminated tumor cells (DTCs) enter a state of dormancy upon arrival at the distant tissue, only to awaken years or even decades afterwards and initiate deadly metastases. How are DTCs kept dormant, what prompts them to awaken, and how can dormant DTCs be targeted are pressing questions in cancer research. Because cancer dormancy provides a unique therapeutic window for preventing metastatic disease, a comprehensive understanding of the distribution, composition and dynamics of reservoirs of dormant DTCs is imperative. Recently, we developed a tool to follow dormant DTCs, and surveyed the anatomical distribution of dormant reservoirs in models of breast cancer metastasis. We discovered that DTCs laid preferentially dormant in the liver, and spatially distinct liver sub-microenvironments allow coexistence of dormant DTCs and rare growing metastases even within the same tissue. To explore whether the distinct fates of DTCs stemmed from intra-organ microenvironmental differences, we performed global transcriptomic profiling on hepatic milieus corresponding to dormancy and metastasis. We found that stromal cells clustered on the basis of disease stage, and genes that were upregulated in dormancy samples were associated with host defense processes and natural killer (NK) cell-mediated response. A thorough examination of viable populations of immune cells in each hepatic milieu revealed that NK cells were the only type of immune cell that increased in dormancy milieus. Gain- and loss-of-function experiments in vivo further indicated that the pool of NK cells encountered by DTCs is decisive on their awakening. Remarkably, adjuvant interleukin-15-based immunotherapy ensured an abundant pool of NK cells that sustained dormancy, thereby preventing hepatic metastases and prolonging survival. In addition, we demonstrated in vivo and in liver-like co-cultures that NK cells control dormant DTCs through IFN-γ-induced quiescence. Thus, we uncovered an alternative cytostatic IFNγ-mediated mechanism of NK cell immunity that is essential to the control of breast cancer dormancy. This finding reroutes the importance of IFNγ to much earlier stages of disease, and encourages its therapeutic use for establishing the non-permissive cytokine milieu that is necessary to limit the emergence of DTCs from dormancy. Another key finding of our work is that liver injury limits NK cell expansion, thus triggering the switch from dormancy to liver metastasis. Using a model of chemically-induced liver injury, we found that activated hepatic stellate cells (aHSCs, which activate from quiescent HSCs to proliferative myofibroblasts) orchestrate a stromal response that hampers NK cell-mediated immunity and precipitates metastasis. Mechanistically, we show that aHSCs secrete the chemokine CXCL12, inducing NK cell quiescence via its cognate receptor CXCR4. This NK cell-inhibitory function of CXCL12 adds to its canonical effect as an inducer of DTC proliferation, and contributes to metastatic outgrowth. Our findings revive clinical interest on the use of CXCR4 inhibitors in the treatment of patients with cancer, but suggest that they should be repurposed to earlier stages to prevent progression of dormant disease. Because the size of the NK cell pool can itself determine metastatic outgrowth, a decrease of NK cells in patients with cancer who are apparently disease-free might identify individuals who are at risk of recurrence and who would benefit from CXCR4 inhibition therapy - a question that we will address in a clinical trial that we are currently setting up at the University Hospital Basel. All this work, which I am the first and corresponding author of, was recently published in Nature (Correia et al. Nature, 2021). In summary, our study shows that DTC dormancy is achieved by preserving normal tissue physiology—particularly tissue innate immunity—and that disruptions of the latter present DTCs with an opportunity for reactivation. In patients, this is reflected in the invariable decline in host defense capabilities that occurs during ageing, but also in the increased risk that recurrent infections and lifestyle-related injury triggers (such as alcohol, obesity and smoking) bring to the sprouting of site-specific metastases. We envision adjuvant NK cell immunotherapy as a means to preserve tissue physiology and prevent metastatic disease. Citation Format: Ana Luisa Correia. Revisiting NK cell immunity to prevent metastasis [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 SY31-02.

Abstract 2450: Investigating the intrinsic and extrinsic factors regulating breast cancer dormancy

Abstract Unfortunately, breast cancer can recur in patients upwards of 25 years after an initial diagnosis and “cure”. It was once thought that recurrence occurred within patients that harbored dormant breast disseminated tumor cells (DTCs) in distant organs that were not present in patients that did not recur. Interestingly, a significant percent of patients harbor DTCs, yet many do not recur, raising the critical question, what differentiates patients that experience a recurrence versus those who live with dormant DTCs for the rest of their lives? It is likely the answer lies in both cell intrinsic as well as extrinsic factors. Nevertheless, the extreme rarity of dormant DTCs has been the major obstacle to their study. To overcome this challenge, we developed an efficient system to isolate and study rare dormant tumor cells from metastatic organs. Using this system and single cell RNA-sequencing (scRNA-seq), we identified a group of genes differentially expressed in dormant breast cancer cells present in both bone and lung. While modulation of these genes individually had no impact on the metastatic behavior of breast cancer cells, we found that as a group, these genes predicted the dormancy phenotype in murine breast cancer models. Importantly, expression of these genes in primary human breast cancer tumors correlated with disease-free survival, suggesting these genes have predictive value in determining which patients are likely to recur. Moreover, we explored extrinsic mechanisms that impact dormancy and found that the chemotherapy reagent doxorubicin (Doxo) drastically changed the microenvironment in vivo, which allowed dormant breast cancer cells to grow robustly within the visceral fat and this was further exacerbated by a high fat diet. Further, we found that Doxo treatment induces significant adipose tissue dystrophy and macrophage infiltration, which may contribute to dormant metastatic outgrowth. Our study provides novel insight into breast cancer dormancy, and also reveals microenvironmental changes that impact dormant breast cancer cell outgrowth. Citation Format: Qihao Ren, Weng Hua Khoo, Jiayu Ye, Douglas V. Faget, Peter I. Croucher, Sheila A. Stewart. Investigating the intrinsic and extrinsic factors regulating breast cancer dormancy [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 2450.

Abstract 916: Dec2, a circadian rhythm gene is necessary for disseminated tumor cell survival in a novel murine model of pancreatic cancer dormancy

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with a proclivity for early metastasis. Resection remains the only chance for long term survival though most patients die from metastatic recurrence. While most patients recur within the first two years, many develop recurrence much later indicating they harbor dormant disseminated tumor cells (dDTCs). Currently there are no murine models which recapitulate this disease pattern and as a result pancreatic cancer dormancy is poorly understood. We sought to better understand PDAC dormancy using a novel murine model of human pancreatic cancer from which we investigate the genetic and molecular contributions to dormancy. A model of resected PDAC was developed using murine PDAC cells expressing luciferase/mCherry orthotopically implanted into FVB hosts. Primary tumors are resected after 4 weeks and mice followed for recurrence. dDTCs’ are harvested from the livers of mice without evidence of recurrence by fluorescence activated cell sorting (FACS) and subjected to transcriptomic profiling using single cell RNA sequencing. Dec2’s contribution to dormancy was evaluated by overexpression and knockout using CRISPR. Overall survival in the mouse model mirrored 41,552 stage I, II PDAC patients from the National Cancer Database with similar frequency and location of recurrence. One third of mice exhibited latent recurrence with median survival of 568 days before succumbing to recurrence. These mice were used to study dDTCs. Transcriptomic profiling of FACS sorted dDTCs revealed a distinct transcriptome from primary tumors and early recurrences, indicating cell plasticity. dDTCs exhibited decreased proliferation markers and upregulated genes involved in immune modulation, cell stemness, linoleic acid (LA) metabolism, and Dec2. dDTCs were resistant to chemotherapy. An in vitro model of dormancy was constructed in which LA treatment inhibited cell proliferation and increased Dec2. Dec2 overexpression resulted in cell quiescence, and knockout increased apoptosis with LA exposure. Dec2-KO cells implanted in the murine model dramatically improved survival compared to Dec-WT cells (Dec2-WT median survival 30 days, Dec2-KO median survival was not reached, p=0.013). Livers of mice in the resection model where Dec2-KO cells were used had decreased dDTC burden with fewer mice showing detectable luciferase gDNA (p<0.01) indicating that loss of Dec2 reduced survival of dDTC’s. This is the first murine model of pancreatic cancer dormancy that recapitulates outcomes of resected PDAC patients. These studies reveal pancreatic cancer dormancy is characterized by a distinct, plastic cellular state characterized by chemotherapy resistance. Dec2 appears required for dormancy, its absence leads to disseminated tumor cell apoptosis. This suggests Dec2 may be a therapeutic target in pancreatic cancer dormancy. Citation Format: Anthony S. Casabianca, Chris Harris, Crissy Dudgeon, Darren R. Carpizo. Dec2, a circadian rhythm gene is necessary for disseminated tumor cell survival in a novel murine model of pancreatic cancer dormancy [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 916.

Feed‐forward loops between metastatic cancer cells and their microenvironment—the stage of escalation

Breast cancer is the most frequent cancer among women, and metastases in distant organs are the leading cause of the cancer‐related deaths. While survival of early‐stage breast cancer patients has increased dramatically, the 5‐year survival rate of metastatic patients has barely improved in the last 20 years. Metastases can arise up to decades after primary tumor resection, hinting at microenvironmental factors influencing the sudden outgrowth of disseminated tumor cells (DTCs). This review summarizes how the environment of the most common metastatic sites (lung, liver, bone, brain) is influenced by the primary tumor and by the varying dormancy of DTCs, with a special focus on how established metastases persist and grow in distant organs due to feed‐forward loops (FFLs). We discuss in detail the importance of FFL of cancer cells with their microenvironment including the secretome, interaction with specialized tissue‐specific cells, nutrients/metabolites, and that novel therapies should target not only the cancer cells but also the tumor microenvironment, which are thick as thieves.

Tracking dormant disseminated tumour cells

Tracking dormant disseminated tumour cells

A tumor-derived type III collagen-rich ECM niche regulates tumor cell dormancy.

Cancer cells disseminate and seed in distant organs, where they can remain dormant for many years before forming clinically detectable metastases. Here we studied how disseminated tumor cells (DTCs) sense and remodel the extracellular matrix (ECM) to sustain dormancy. ECM proteomics revealed that dormant cancer cells assemble type III collagen-enriched ECM niche. Tumor-derived type III collagen is required to sustain tumor dormancy as its disruption restores tumor cell proliferation through DDR1-mediated STAT1 signaling. Second harmonic generation two-photon microscopy further revealed that the dormancy-to-reactivation transition is accompanied by changes in type III collagen architecture and abundance. Analysis of clinical samples revealed that type III collagen levels were increased in tumors from lymph node-negative head and neck squamous cell carcinoma (HNSCC) patients compared to lymph node-positive patients. Our data supports that the manipulation of these mechanisms could serve as a barrier to metastasis through DTCs dormancy induction.

Transcriptomic Profiling Reveals Novel Candidate Genes and Signalling Programs in Breast Cancer Quiescence and Dormancy.

Simple SummaryBreast cancer can return many years after treatment of the primary tumour. This is caused by cells that have spread to other parts of the body and entered a non-dividing state called quiescence or dormancy that can last for decades. Dormant cancer cells are not sensitive to conventional chemo- and radiotherapies, which primarily target fast growing cells, and so can eventually resume growth to cause formation of tumours at secondary sites. The exact processes by which cancer cells become dormant are currently poorly understood. This study describes the use of model systems specifically developed to compare the genes used by dormant and dividing breast cancer cells, allowing the identification of a number of genes and cellular mechanisms that might underpin breast cancer cell dormancy and therefore represent promising novel candidates to inform development of new treatments to prevent breast cancer recurrence.Metastatic recurrence, the major cause of breast cancer mortality, is driven by reactivation of dormant disseminated tumour cells that are defined by mitotic quiescence and chemoresistance. The molecular mechanisms underpinning mitotic quiescence in cancer are poorly understood, severely limiting the development of novel therapies for removal of residual, metastasis-initiating tumour cells. Here, we present a molecular portrait of the quiescent breast cancer cell transcriptome across the four main breast cancer sub-types (luminal, HER2-enriched, basal-like and claudin-low) and identify a novel quiescence-associated 22-gene signature using an established lipophilic-dye (Vybrant® DiD) retention model and whole-transcriptomic profiling (mRNA-Seq). Using functional association network analysis, we elucidate the molecular interactors of these signature genes. We then go on to demonstrate that our novel 22-gene signature strongly correlates with low tumoural proliferative activity, and with dormant disease and late metastatic recurrence (≥5 years after primary tumour diagnosis) in metastatic breast cancer in multiple clinical cohorts. These genes may govern the formation and persistence of disseminated tumour cell populations responsible for breast cancer recurrence, and therefore represent prospective novel candidates to inform future development of therapeutic strategies to target disseminated tumour cells in breast cancer, eliminate minimal residual disease and prevent metastatic recurrence.

Thorny ground, rocky soil: Tissue-specific mechanisms of tumor dormancy and relapse.

Disseminated tumor cells (DTCs) spread systemically yet distinct patterns of metastasis indicate a range of tissue susceptibility to metastatic colonization. Distinctions between permissive and suppressive tissues are still being elucidated at cellular and molecular levels. Although there is a growing appreciation for the role of the microenvironment in regulating metastatic success, we have a limited understanding of how diverse tissues regulate DTC dormancy, the state of reversible quiescence and subsequent awakening thought to contribute to delayed relapse. Several themes of microenvironmental regulation of dormancy are beginning to emerge, including vascular association, co-option of pre-existing niches, metabolic adaptation, and immune evasion, with tissue-specific nuances. Conversely, DTC awakening is often associated with injury or inflammation-induced activation of the stroma, promoting a proliferative environment with DTCs following suit. We review what is known about tissue-specific regulation of tumor dormancy on a tissue-by-tissue basis, profiling major metastatic organs including the bone, lung, brain, liver, and lymph node. An aerial view of the barriers to metastatic growth may reveal common targets and dependencies to inform the therapeutic prevention of relapse.

Innovative Approaches in the Battle Against Cancer Recurrence: Novel Strategies to Combat Dormant Disseminated Tumor Cells.

Cancer recurrence remains a great fear for many cancer survivors following their initial, apparently successful, therapy. Despite significant improvement in the overall survival of many types of cancer, metastasis accounts for ~90% of all cancer mortality. There is a growing understanding that future therapeutic practices must accommodate this unmet medical need in preventing metastatic recurrence. Accumulating evidence supports dormant disseminated tumor cells (DTCs) as a source of cancer recurrence and recognizes the need for novel strategies to target these tumor cells. This review presents strategies to target dormant quiescent DTCs that reside at secondary sites. These strategies aim to prevent recurrence by maintaining dormant DTCs at bay, or eradicating them. Various approaches are presented, including: reinforcing the niche where dormant DTCs reside in order to keep dormant DTCs at bay; promoting cell intrinsic mechanisms to induce dormancy; preventing the engagement of dormant DTCs with their supportive niche in order to prevent their reactivation; targeting cell-intrinsic mechanisms mediating long-term survival of dormant DTCs; sensitizing dormant DTCs to chemotherapy treatments; and, inhibiting the immune evasion of dormant DTCs, leading to their demise. Various therapeutic approaches, some of which utilize drugs that are already approved, or have been tested in clinical trials and may be considered for repurposing, will be discussed. In addition, clinical evidence for the presence of dormant DTCs will be reviewed, along with potential prognostic biomarkers to enable the identification and stratification of patients who are at high risk of recurrence, and who could benefit from novel dormant DTCs targeting therapies. Finally, we will address the shortcomings of current trial designs for determining activity against dormant DTCs and provide novel approaches.

Prognosis of patients treated in a single neurosurgical reference centre for brain metastasis caused by dormant disseminated cells.

Brain metastasis (BM) is a frequent complication of systemic cancer usually associated with poor prognosis. Survival depends on numerous factors, which complicates prognosis and treatment. It has been suggested that BM growing from previously dormant disseminated tumour cells (DTCs) may exhibit a milder phenotype than BM derived from continuously progressing metastatic cells; however, to the best of our knowledge, the prognosis of patients presenting with BM from dormant DTCs is unknown. The present study retrospectively compared survival data, collected from a single neurosurgical centre, between patients presenting with BM from previously dormant DTCs and patients with non-dormant BM. A total of 262 medical records were reviewed. In the univariate Cox regression analysis, the median survival of the dormant BM group was statistically longer than that of the non-dormant group (P=0.048); a trend towards a longer survival persisted after correcting for age, presence of breast cancer and treatment options (P=0.057), which are all factors known to influence outcome. The improved outcome of these patients could be considered in models for prognostication. Moreover, the development of therapies able to eradicate dormant DTCs could provide a new promising strategy to prolong the survival of patients with a favourable prognosis.

Targeting Intercellular Communication in the Bone Microenvironment to Prevent Disseminated Tumor Cell Escape from Dormancy and Bone Metastatic Tumor Growth.

Metastasis to the bone is a common feature of many cancers including those of the breast, prostate, lung, thyroid and kidney. Once tumors metastasize to the bone, they are essentially incurable. Bone metastasis is a complex process involving not only intravasation of tumor cells from the primary tumor into circulation, but extravasation from circulation into the bone where they meet an environment that is generally suppressive of their growth. The bone microenvironment can inhibit the growth of disseminated tumor cells (DTC) by inducing dormancy of the DTC directly and later on following formation of a micrometastatic tumour mass by inhibiting metastatic processes including angiogenesis, bone remodeling and immunosuppressive cell functions. In this review we will highlight some of the mechanisms mediating DTC dormancy and the complex relationships which occur between tumor cells and bone resident cells in the bone metastatic microenvironment. These inter-cellular interactions may be important targets to consider for development of novel effective therapies for the prevention or treatment of bone metastases.

Abstract PO005: Hepatic stellate cells suppress NK cell sustained breast cancer dormancy

Abstract The persistence of undetectable disseminated tumour cells (DTCs) after primary tumour resection poses a major challenge to effective cancer treatment. These enduring dormant DTCs are seeds of future metastases, and the mechanisms that switch them from dormancy to outgrowth require definition. Because cancer dormancy provides a singular therapeutic window to prevent metastatic disease, a comprehensive understanding of the distribution, composition and dynamics of dormant DTC reservoirs is imperative. Here we reveal tissue-specific microenvironments that restrain or concede progression of breast cancer in the liver, a frequent site of metastasis and most times associated with patients’ cause of death. Using mouse models, we show that the dormant milieu features a selective accumulation of natural killer (NK) cells. Preserving the NK cell pool with adjuvant interleukin-15-based immunotherapy prevents hepatic metastases and prolongs survival. Exit from dormancy follows a dramatic contraction of the NK cell compartment, and concurrent accumulation of activated stellate cells. Our data identify the interplay between NK cells and activated stellate cells as a master switch of cancer dormancy, and suggest that therapies aimed at normalizing the NK cell pool might succeed in preventing DTC outgrowth and metastasis. Citation Format: Ana Luisa Correia. Hepatic stellate cells suppress NK cell sustained breast cancer dormancy [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO005.

Abstract PD9-11: Identifying breast cancer survivors with dormant disseminated tumor cells: The PENN-SURMOUNT screening study

Abstract Background: Patients (pts) treated for early stage breast cancer have a 30% lifetime risk of developing incurable, distant metastatic disease. Current models suggest that this occurs through escape of cells from the primary tumor into the circulation and subsequent sequestration of “disseminated tumor cells” (DTCs), in the bone marrow and other sequestration sites, where they enter dormancy. DTCs identified by immunohistochemistry (IHC) are associated with poor prognosis in longitudinal studies and meta-analyses, increasing odds of recurrence by approximately 2 to 5-fold. However, little is known about the test characteristics of the DTC-IHC assay, clinical DTC detection rates over time, and patient and disease risk factors that can identify pts harboring these cells. Methods: The PENN-SURMOUNT Screening Study (NCT 02732171) is a prospective, longitudinal study examining bone marrow and blood biomarkers of recurrence among pts within 5 years of diagnosis who have completed therapy for primary breast cancer (with the exception of endocrine therapy). Pts with positive lymph nodes, triple negative receptors, ER-positivity with RS ≥ 25 and/or high-risk MammaPrint (MP), or residual disease after neoadjuvant chemotherapy were screened with bone marrow aspirate (BMA) for presence of DTCs. A positive DTC-IHC result is defined by the presence of at least one pancytokeratin-DAB positive cell utilizing the methods of Naume et al. Cytospin slides prepared from the BMA are independently reviewed by two pathologists with adjudication for the presence of DTCs; Pts who screen negative for DTCs are offered repeat screening annually. Pts who screen positive are referred to an interventional clinical trial (CLEVER, NCT 03032406). Results: A total of 194 pts screened eligible for enrollment on PENN-SURMOUNT between 6/2016 and 3/2020. Of these, 158 consented and 151 underwent BMA with successful IHC analysis on 100%. Pts came from 22 U.S. states; ≥ 1/3 traveled over 50 miles to the study center. At baseline BMA, 36/151 (24%) had at least 1 measurable DTC by IHC. Patient characteristics and DTC distribution among subpopulations are shown in Table 1. Of the 78/115 who were initially DTC negative and continued to be eligible for repeat screening, as of 3/2020, 46 (59%) returned for at least one repeat BMA. 13/46 (28%) had at least 1 detectable DTC on 1 of up to 3 subsequent follow up assessments for a total DTC positivity rate of 32.5% (49/151). 48 (98%) DTC+ pts have subsequently enrolled on the CLEVER trial. Conclusions: BMA assessment for DTCs is feasible in pts with high risk, early stage breast cancer. DTCs are detected in up to a third of breast cancer survivors with repeat assessment during the surveilance period. DTC positivity rates are relatively similar across all receptor subtypes, and after both neoadjuvant and adjuvant chemotherapy. Pts harboring DTCs are highly likely to enroll on interventional trials designed to reduce recurrence risk. Table 1. Patient characteristics and distribution of % DTC positivity among subpopulationsDTC+ (N=49)DTC- (N=102)Total (N=151)DTC+ Rate (Overall: 32.5%)DEMOGRAPHICSMedian Age at BMA (yrs)51.9 (43.9-60.6*)50.5 (42.9-58.1)50.5 (43.8-58.8)N/ARaceCaucasian44 (89.8%)91 (89.2%)135 (89.4%)32.6%African American5 (10.2%)9 (8.8%)14 (9.3%)35.7%Other0 (0%)2 (2.0%)2 (1.3%)0%Menopausal StatusPre-15 (30.6%)34 (33.3%)49 (32.5%)30.6%Post-34 (69.4%)68 (66.7%)102 (67.5%)33.3%BMI at BMA (kg/m2)24.2 (21.9-28.9*)26.9 (23.4-31.4)26.1 (22.8-30.4)N/ARECEPTOR STATUSER/PR+ HER2neg (by ASCO/CAP)24 (49.0%)51 (50.0%)75 (49.7%)32.0%HER2+ (any ER/PR)9 (18.4%)12 (11.8%)21 (13.9%)42.9%ER/PRneg HER2neg23 (46.9%)48 (47.1%)71 (47.0%)32.4%RISK CRITERIALymph Node Positive24 (49.0%)65 (63.7%)89 (58.9%)27.0%Triple Negative (ER/PR<10%)27 (55.1%)50 (49.0%)77 (51.0%)35.0%Non-pCR11 (22.4%)25 (24.5%)36 (23.8%)30.6%RS ≥ 25 and/or High Risk MP6 (12.2%)8 (7.8%)14 (9.3%)42.9%Median T size (cm) -excluding NACT2.1 (1.5-2.9*)1.8 (1.3-2.8)1.8 (1.3-2.9)N/APRIOR THERAPYAdjuvant Chemo25 (51.0%)60 (58.8%)85 (56.3%)29.4%Neoadjuvant Chemo22 (44.9%)41 (40.2%)63 (41.7%)34.9%Endocrine Therapy19 (38.8%)47 (46.1%)66 (43.7%)28.8%XRT29 (59.2%)75 (73.5%)104 (69.3%^)27.9%*Ranges represent interquartile range^ XRT data not available on 1 patient; n=150 was used to figure percentage Citation Format: Lauren Bayne, Isoris Nivar, Brooke Goodspeed, Shannon Deluca, E. Paul Wileyto, Natalie Shih, Anupma Nayak, Michael D Feldman, Joshua Edwards, Kevin Fox, Jennifer M. Matro, Susan Domchek, Hayley Knollman, Rachel Jankowitz, Angela Bradbury, Payal D. Shah, Jewell Graves, George Woodfield, Elizabeth Chislock, Jianping Wang, George Belka, Lewis A. Chodosh, Amy S. Clark, Angela DeMichele. Identifying breast cancer survivors with dormant disseminated tumor cells: The PENN-SURMOUNT screening study [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD9-11.