Dormant Cancer Research Articles

EBV promotes vascular mimicry of dormant cancer cells by potentiating stemness and EMT

Vascular mimicry (VM) is defined as a vascular channel-like structure composed of tumor cells that correlates with the growth of cancer cells by providing blood circulation. However, whether VM can be formed in dormant cancer cells remains unclear. Our previous research revealed that polyploid giant cancer cells (PGCCs) are specific dormant cells related to the poor prognosis of head and neck cancer. Here, we demonstrated that EBV could promote VM formation by PGCCs in vivo and in vitro. Furthermore, we revealed that the activation of the ERK pathway partly mediated by LMP2A is responsible for stemness, and the acquisition of the stemness phenotype is crucial to the malignant biological behavior of PGCCs. The epithelial-to-mesenchymal transition (EMT) process plays a considerable role in PGCCs, and EMT progression is vital for EBV-positive PGCCs to form VM. This is the first study to reveal that EBV creates plasticity in PGCC-VM and provide a new strategy for targeted anti-tumor therapy.

Phenotypic Characteristics of Dormant Human Non-Small Cell Lung Cancer Cells Surviving Multifraction X-Ray Irradiation.

Phenotypic characteristics of human non-small cell lung cancer cells, A549 (p53 wild-type) and H1299 (p53-deficient) as well as their descendants surviving after multifraction X-ray irradiation at a cumulative dose of 60 Gy (sublines A549HR and H1299HR, respectively) were studied before and after additional 2 Gy single dose irradiation. In 24 h after the additional irradiation, we observed a significant increase in the proportion of cells with signs of entosis (by 5 times, p<0.05) and SA-β-gal+ cells (by 1.6 times, p<0.01) in the general population of A549HR cells. In contrast, a significant increase in the proportion of only SA-β-gal+ multinucleated giant cancer cells was revealed in the parental A549 cells. Additional single dose irradiation resulted in a significant (by 1.8 times, p<0.05) increase in the proportion of multinucleated giant cancer cells in H1299HR cells in comparison with their parental H1299 cells. These changes did not correlate with changes in the proportion of entotic cells, because their high basal content in the absence of functional p53 did not change in response to additional single dose irradiation. At the same time, both p53-deficient non-small cell lung cancer cell lines showed a significant (2.9-fold for H1299 and 5.5-fold for H1299HR cells, p<0.001) increase in the proportion of SA-β-gal+ cells in the general population, but not in the multinucleated giant cancer cells population.

Can 3D bioprinting solve the mystery of senescence in cancer therapy?

Tumor dormancy leading to cancer relapse is still a poorly understood mechanism. Several cell states such as quiescence and diapause can explain the persistence of tumor cells in a dormant state, but the potential role of tumor cell senescence has been met with hesitance given the historical understanding of the senescent growth arrest as irreversible. However, recent evidence has suggested that senescence might contribute to dormancy and relapse, although its exact role is not fully developed. This limited understanding is largely due to the paucity of reliable study models. The current 2D cell modeling is overly simplistic and lacks the appropriate representation of the interactions between tumor cells (senescent or non-senescent) and the other cell types within the tumor microenvironment (TME), as well as with the extracellular matrix (ECM). 3D cell culture models, including 3D bioprinting techniques, offer a promising approach to better recapitulate the native cancer microenvironment and would significantly improve our understanding of cancer biology and cellular response to treatment, particularly Therapy-Induced Senescence (TIS), and its contribution to tumor dormancy and cancer recurrence. Fabricating a novel 3D bioprinted model offers excellent opportunities to investigate both the role of TIS in tumor dormancy and the utility of senolytics (drugs that selectively eliminate senescent cells) in targeting dormant cancer cells and mitigating the risk for resurgence. In this review, we discuss literature on the possible contribution of TIS in tumor dormancy, provide examples on the current 3D models of senescence, and propose a novel 3D model to investigate the ultimate role of TIS in mediating overall response to therapy.

Modeling ER+ breast cancer dormancy in mice

Modeling ER+ breast cancer dormancy in mice

Biological effects of COVID-19 on lung cancer: Can we drive our decisions.

COVID-19 infection caused by SARS-CoV-2 is considered catastrophic because it affects multiple organs, particularly those of the respiratory tract. Although the consequences of this infection are not fully clear, it causes damage to the lungs, the cardiovascular and nervous systems, and other organs, subsequently inducing organ failure. In particular, the effects of SARS-CoV-2-induced inflammation on cancer cells and the tumor microenvironment need to be investigated. COVID-19 may alter the tumor microenvironment, promoting cancer cell proliferation and dormant cancer cell (DCC) reawakening. DCCs reawakened upon infection with SARS-CoV-2 can populate the premetastatic niche in the lungs and other organs, leading to tumor dissemination. DCC reawakening and consequent neutrophil and monocyte/macrophage activation with an uncontrolled cascade of pro-inflammatory cytokines are the most severe clinical effects of COVID-19. Moreover, neutrophil extracellular traps have been demonstrated to activate the dissemination of premetastatic cells into the lungs. Further studies are warranted to better define the roles of COVID-19 in inflammation as well as in tumor development and tumor cell metastasis; the results of these studies will aid in the development of further targeted therapies, both for cancer prevention and the treatment of patients with COVID-19.

Leveraging the scientific findings to develop therapeutic strategies for dormant breast cancer cells.

Breast cancer (BC) metastasis can occur decades before clinical diagnosis. During this time, the cancer cells (BCCs) can remain dormant for decades. This type of dormancy also occurs during remission where the dormant BCCs adapt cycling quiescence within the tissue microenvironment. BC shows preference for the bone marrow (BM), resulting in poor prognosis. The BM provides a challenge due to the complex niche between the peripheral interface and endosteum. The process of dormancy begins upon entry into the marrow with the changes facilitated through crosstalk between the cancer cells and tissue niche. More importantly, dormancy can occur at any time during the disease process, including the time during treatment. This perspective discusses the challenges posed by the marrow microenvironment to develop treatment. The article discusses the complex mechanisms at each compartment within the marrow niche and the added negative issue of toxicity to the endogenous stem cells.

Autophagy promotes recurrence of nasopharyngeal carcinoma via inducing the formation of dormant polyploid giant cancer cells

Objective: To explore the effect of dormant polyploid giant cancer cells (PGCC) on nasopharyngeal carcinoma (NPC) recurrence and to clarify the role of inhibition of autophagy in inhibiting NPC-PGCC formation and preventing NPC recurrence. Methods: NPC cells-derived PGCC (NPC-PGCC) were induced by paclitaxel (PTX), and the morphology, polyploid characteristics and cell activity of PGCC were identified by light microscopy, immunofluorescence and Live/Dead cell double staining assays. RNA-seq was used to analyze the differentially expressed genes between NPC-PGCC and diploid nasopharyngeal carcinoma cells CNE2. Functional enrichment and pathway annotation analysis of differentially expressed genes were performed using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG). The level of autophagy in NPC-PGCC cells was assessed by Western Blot and transmission electron microscopy analysis. The role of autophagy in the formation of NPC-PGCC and the effect of NPC-PGCC on the recurrence of nasopharyngeal carcinoma were studied using a highly clinically relevant mouse nasopharyngeal carcinoma recurrence model. Statistical analysis was performed using GraphPad Prism 6 and P-values<0.05 were considered statistically significant. Results: NPC-PGCC induced by paclitaxel had the characteristics of burst-like division after dormancy. GO enrichment and KEGG pathway analyses identified the significant biological processes and pathways mainly concentrated in autophagy and related pathways involving the differentially expressed genes between NPC-PGCC and diploid nasopharyngeal carcinoma cells CNE2. The autophagy level was significantly enhanced in NPC-PGCC cells. In a highly clinically relevant mouse nasopharyngeal carcinoma recurrence model, the number of PGCC in the primary tumor of the nude mice treated with cisplatin were higher than those of the other groups. In nude mice pretreated with autophagy inhibitor and then co-treatment with autophagy inhibitor and cisplatin, the number of PGCC in primary tumors was less and the recurrence rate was significantly lower than in other groups. Conclusions: The mechanism of dormant polyploid giant cancer cells formation is related to autophagy. Inhibition of autophagy can inhibit the formation of PGCC and thus prevent the recurrence of nasopharyngeal carcinoma.

The role of the bone microenvironment in regulating myeloma residual disease and treatment.

Multiple myeloma is an incurable haematological cancer. The increase in targeted therapies has improved the number of myeloma patients achieving a complete response and improved progression-free survival following therapy. However, a low level of disease or minimal residual disease (MRD) still persists which contributes to the inevitable relapse in myeloma patients. MRD has been attributed to the presence of dormant myeloma cells and their subsequent reactivation, which is controlled by the microenvironment and specialised niches within the bone marrow. This contributes to the evasion of the immune system and chemotherapy, eventually leading to relapse. The growth of myeloma tumours are heavily dependent on environmental stimuli from the bone marrow microenvironment, and this plays a key role in myeloma progression. The bone microenvironment also plays a critical role in myeloma bone disease and the development of skeletal-related events. This review focuses on the bone marrow microenvironment in relation to myeloma pathogenesis and cancer dormancy. Moreover, it reviews the current therapies targeting the bone microenvironment to treat myeloma and myeloma bone disease. Lastly, it identifies novel therapeutic targets for myeloma treatment and the associated bone disease.

Stromal Co-Cultivation for Modeling Breast Cancer Dormancy in the Bone Marrow.

Simple SummaryCancer cells travel to far away parts of the body before the original cancer can be detected. Breast cancer cells travel mainly to the bone marrow, where they are kept in a quiet, non-dividing, dormant state for many years by cells and proteins in the bone marrow. These metastatic cancer cells wake up at a steady rate over the years and result in incurable disease. This paper outlines how to study the interactions of cells that live in the bone marrow with cancer cells in the laboratory. We describe how to grow mixtures of a variety of bone marrow-residing cells to generate cell layers upon which cancer cells can be grown. Using this system, we can study their effects on cancer cell growth or dormancy and the biology of the interactions. With this approach, we can study how to either eliminate the cancer cells or prevent them from waking up.Cancers metastasize to the bone marrow before primary tumors can be detected. Bone marrow micrometastases are resistant to therapy, and while they are able to remain dormant for decades, they recur steadily and result in incurable metastatic disease. The bone marrow microenvironment maintains the dormancy and chemoresistance of micrometastases through interactions with multiple cell types and through structural and soluble factors. Modeling dormancy in vitro can identify the mechanisms of these interactions. Modeling also identifies mechanisms able to disrupt these interactions or define novel interactions that promote the reawakening of dormant cells. The in vitro modeling of the interactions of cancer cells with various bone marrow elements can generate hypotheses on the mechanisms that control dormancy, treatment resistance and reawakening in vivo. These hypotheses can guide in vivo murine experiments that have high probabilities of succeeding in order to verify in vitro findings while minimizing the use of animals in experiments. This review outlines the existing data on predominant stromal cell types and their use in 2D co-cultures with cancer cells.

Clinical and Biological Aspects of Disseminated Tumor Cells and Dormancy in Breast Cancer.

Progress in detection and treatment have drastically improved survival for early breast cancer patients. However, distant recurrence causes high mortality and is typically considered incurable. Cancer dissemination occurs via circulating tumor cells (CTCs) and up to 75% of breast cancer patients could harbor micrometastatses at time of diagnosis, while metastatic recurrence often occurs years to decades after treatment. During clinical latency, disseminated tumor cells (DTCs) can enter a state of cell cycle arrest or dormancy at distant sites, and are likely shielded from immune detection and treatment. While this is a challenge, it can also be seen as an outstanding opportunity to target dormant DTCs on time, before their transformation into lethal macrometastatic lesions. Here, we review and discuss progress made in our understanding of DTC and dormancy biology in breast cancer. Strides in our mechanistic insights of these features has led to the identification of possible targeting strategies, yet, their integration into clinical trial design is still uncertain. Incorporating minimally invasive liquid biopsies and rationally designed adjuvant therapies, targeting both proliferating and dormant tumor cells, may help to address current challenges and improve precision cancer care.

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 LB025: Malat1-Serpinb6b signaling blocks T cells-mediated pyroptosis and governs cancer dormancy metastatic reactivation

Abstract Metastatic relapse is the major causes of mortality in patients with cancer and occur due to metastatic reactivation of dormant tumor cells. Early dissemination of tumor cells undergoing a protected period of dormancy in the target organs potentially explains this prevalent clinical behavior. Long non-coding RNAs (lncRNAs) are involved in various biological processes and diseases. Malat1 is one of the most abundant and highly conserved nuclear lncRNAs and have shown the associated with metastasis and serving as a predictive marker for various tumor progression. However, the correlation of tumor intrinsic lncRNAs in regulation of tumor dormancy and immune evasion are largely unknown. Using an in vivo screening platform for the isolation of genetic entities involved in either dormancy or reactivation of breast cancer tumor cells, we have identified Malat1 as a positive mediator of metastatic reactivation. To dissect the functional role of Malat1 in metastatic reactivation, we developed a clean Malat1 knockout (KO) model using paired gRNA CRISPR-Cas9 in metastatic murine syngeneic breast cancer. As a proof of concept, we also used inducible knockdown system under in vivo models. To delineate the immune microenvironment, we used single cell RNA-seq, ChIRP-seq, multicolor flowcytometry, RNA-FISH, and coculture experiments. Our data revealed that deletion of Malat1 induces dormancy and attenuated the metastatic colonization resulting in long-term survival of syngeneic mice model. In contrast, overexpression of Malat1 leads to metastatic reactivation of dormant breast cancer cells. Interestingly, 4T1-Malat1 KO dormant breast cancer cells exhibit metastatic outgrowth in T cells defective mice. Our single-cell RNA-seq and multicolor flowcytometry evaluation reveal enhanced T cells and reduced neutrophils proportions in mice with Malat1 KO cells. This indicates a critical role of immune microenvironment via Malat1-dependent immune evasion. Additionally, Malat1 KO inhibits cancer stemness properties. Similarly, RNA-seq and ChIRP-seq data suggest that KO of Malat1 hampers immune evasion and downregulates metastasis associated genes including Serpins and Wnts. Additionally, our data strongly suggests that Malat1 KO cells persists as non-proliferative dormant cells in lung due to CD8+ T cell-umpired immune activity. Our mechanistic studies showed that Malat1 regulated Sepinb6b blocks the CstG enzymatic activity and suppress cleavage of GsdmD and T cells induced pyroptosis. Interestingly, rescue experiments suggest that Malat1 or Serpinb6b protects T cell-induced cell death and induces dormancy re-awakening thereby rescue the metastatic potential of 4T1 Malat1 KO cells. Combination of Malat1 ASO with double immune checkpoint inhibitors greatly affects the metastatic outgrowth in breast cancer. Taken together, our studies demonstrate that tumor intrinsic Malat1 regulates Serpinb6b that suppresses CstG-GsdmD mediated pyroptosis which eventually promotes immune evasion and breast cancer dormancy metastatic reactivation. Citation Format: Dhiraj Kumar, Filippo Giancotti. Malat1-Serpinb6b signaling blocks T cells-mediated pyroptosis and governs cancer dormancy metastatic reactivation [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 LB025.

Abstract SY31-01: Tissue resident macrophages and a mesenchymal-like program control early disseminated cancer cell dormancy

Abstract Tissue resident macrophages and a mesenchymal-like program control early disseminated cancer cell dormancy. Julio A. Aguirre-GhisoDepartment of Cell Biology, Department of Medicine-Oncology, Cancer Dormancy and Tumor Microenvironment Institute, Albert Einstein Cancer Center, Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA. Increasing evidence shows that cancer cells can disseminate from early-evolved primary lesions much earlier than the classical metastasis models predicted. The current paradigm supports that the tissue microenvironment where disseminated cancer cells (DCCs) lodge is a critical determinant of the timing of metastasis. However, how tissue-resident myeloid cells control this process is largely unclear. The lung tissue contains a resident population of alveolar macrophages (AMs), whose function in metastatic dormancy and growth is largely unexplored. Here, we used various transgenic mouse models, human sample analysis and single cell RNA-sequencing to reveal DCC heterogeneity and plasticity in the lung across disease evolution. We found a previously unrecognized role of mesenchymal- and pluripotency-like (M-like) programs driven by the transcription factor ZFP281 in coordinating early cancer cell spread and a long-lived dormancy program in early DCCs. The gene profiles revealed molecules that would allow early DCCs to crosstalk with AMs. Using a 3D Matrigel assay, we found that AMs are responsible for inducing an M-like phenotype in cancer cells (of both early and late evolved cancer cells) and exclusively suppressed growth of early lesion (EL) HER2+ cancer cells. The conditioned media (CM) from AMs was also found to induce an M-like phenotype and growth arrest of EL cells. RNA-sequencing profiling of pure EL cells and AMs that had been co-cultured revealed a reciprocal influence of the two cell types: increased ECM organization and induction of invasiveness in EL cells, as well as changes in inflammatory response and immune programs in AMs. Prediction of ligand-receptor interactions using the NATMI tool allowed for identification of signaling axis that would allow AMs to induce an M-like and growth program in EL cells. To test whether indeed AMs are inducing an M-like and growth arrest phenotype in early DCCs in lungs, we depleted AMs in mice bearing early MMTV-HER2 or late evolved E0771 breast cancer cells in lungs via intranasal instillation of clodronate liposomes or via the use of a AM=specific CD169-DTR targeting system. Following AM depletion, dormant DCCs began to proliferate and formed clusters and overt metastases. Our results support that tissue resident macrophages play a key role in limiting metastatic expansion of breast cancer cells via the induction of a DCC mesenchymal-like dormancy program. Citation Format: Julio A. Aguirre-Ghiso. Tissue resident macrophages and a mesenchymal-like program control early disseminated cancer cell 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 SY31-01.

Abstract 1329: Maresins prevent breast cancer dormancy escape via resolution of inflammation

Abstract Cytotoxic cancer therapies reduce tumor burden by killing tumor cells. However, the resulting apoptotic and necrotic cell bodies (tumor cell “debris”) may stimulate tumor initiation and progression by disrupting the resolution of inflammation. Thus, chemotherapy and anti-estrogen breast cancer therapy, including tamoxifen, may be a double-edged sword. A paradigm shift is emerging in understanding the resolution of inflammation as an active biochemical process with the discovery of novel specialized pro-resolving lipid autocoid mediators (SPMs), such as maresins and endogenous resolution programs. Despite approaches to block systemic inflammation, there are no current “pro-resolving” therapies in cancer. To determine whether debris stimulates breast cancer growth, we utilized tumor dormancy models with a subthreshold (nontumorigenic) inoculum of tumor cells. We demonstrated that breast tumor “debris” generated by cytotoxic anti-estrogen therapy (tamoxifen or fulvestrant) or chemotherapy (eribulin) stimulates dormancy escape by triggering a macrophage-derived pro-inflammatory and pro-angiogenic “cytokine storm”. Thus, tumor cell debris is a critical pro-tumorigenic factor in breast cancer initiation and progression. To assess whether stimulating the clearance of debris would suppress breast cancer progression, we utilized the SPMs maresin 1 (MaR1) and maresin conjugates in tissue regeneration (MCTR1, MCTR2). Each maresin (MaR1, MCTR1 and MCTR2) sharply reduced tumor growth in both debris-stimulated and spontaneous (e.g. MMTV-PyMT) breast cancer models at nanogram concentrations (15 ng/day) without toxicity. Notably, maresins enhanced immunotherapy (anti-CTLA4) to induce tumor regression in estrogen receptor (ER) positive (EO771) and inhibit ER negative tumor growth (4T1). Maresins stimulated macrophage phagocytosis of therapy (fulvestrant and tamoxifen)-generated breast cancer debris at only nanomolar concentrations (0.1 - 10 nM). Remarkably, maresins alone or in combination with chemotherapy (paclitaxel) reduced levels of pro-angiogenic factors (e.g. CXCL12/SDF-1) in the tumor microenvironment and decreased microvessel density/size, thereby inhibiting tumor angiogenesis. Maresins dampened the therapy-induced cytokine storm, by reducing levels of TNF-α, MIP-2/CXCL2, CCL2/MCP-1, IL-1ra/IL-1F3, CCL5, CXCL13, Serpin E1/PAI-1, IL-1β and G-CSF both in vitro in debris-stimulated macrophages and in vivo in plasma and tumor tissue. Stimulating the resolution of inflammation via pro-resolution lipid mediators to enhance immunotherapy is a novel host-centric therapeutic approach to prevent breast cancer initiation, dormancy escape and tumor progression via debris clearance and counter-regulation of the cytokine storm. Altogether, the maresin pathway mediators may represent a new therapeutic approach to stimulate the resolution of inflammation in breast cancer. Citation Format: Franciele Cristina Kipper, Jianjun Deng, Eva Rothenberger, Abigail Kelly, Madeline Duncan, Sui Huang, Charles N. Serhan, Dipak Panigrahy. Maresins prevent breast cancer dormancy escape via resolution of inflammation [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 1329.

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 4065: Efficacy of the Rac/Cdc42 inhibitor MBQ-167 in combination therapy with Paclitaxel

Abstract Triple negative breast cancer (TNBC) represents a challenge because of its aggressiveness and resistance to standard chemotherapy. Since TNBC lacks targeted therapy options, the frontline therapy is Paclitaxel, which targets actively dividing cells inducing subsequent apoptosis. Since dormant cells or metastatic cancer cells are not targeted by Paclitaxel, more targeted treatment options are needed. MBQ-167, a small molecule developed by us to target the metastasis drivers Rac and Cdc42, is highly effective in reducing tumor growth and metastasis in mouse models of TNBC (Cruz-Collazo et al., 2021, Molecular Cancer Therapeutics). The purpose of this study is to test the hypothesis that MBQ-167 is a viable candidate for combined therapy with Paclitaxel in TNBC. Therefore, we tested the efficacy of MBQ-167 in combination with Paclitaxel in MDA-MB-231 human TNBC cells via MTT viability assays and caspase 3/7 apoptosis, using effective concentrations of MBQ-167 and/or Paclitaxel for 24, 48, 96 &amp; 120 hrs. We found that individual MBQ-167 or the combination therapy was more effective at 48-120 hrs at reducing cell viability by ~80% compared to ~60% by Paclitaxel treatment alone and significatively increasing apoptosis. Next, we tested individual or combined MBQ-167 (5 mg/kg 5X a week) and Paclitaxel (10 mg/kg 1X a week) on SCID mice bearing MDA-MB-231 tumors, via intraperitoneal administration. We report a significant reduction in tumor growth and lung metastasis in response to combined MBQ-167 and Paclitaxel compared to either treatment alone. A more aggressive syngeneic model of 4T-1 mouse breast cancer cells in BALB/c immunocompetent model was used to determine the direct effect of MBQ-167 and Paclitaxel on metastasis. When 4T-1 tumors were surgically removed and MBQ-167, Paclitaxel, or the combination administered for 17 days, the combination treatment, but not individual compounds, significantly reduced lung metastases by ~80%. In conclusion, this study validates the clinical testing of MBQ-167 in combination with Paclitaxel as a potential therapeutic for TNBC. Citation Format: Ailed M. Cruz-Collazo, Olga Katsara, Elizabeth Salvo, Jean Ruiz, Robert Schneider, Suranganie Dharmawardhane. Efficacy of the Rac/Cdc42 inhibitor MBQ-167 in combination therapy with Paclitaxel [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 4065.

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&amp;lt;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.

Abstract 1544: The lncRNA SNHG1 modulates quiescence and chemoresistance of prostate cancer

Abstract Prostate cancer dormancy and chemoresistance contributes to long term morbidity and mortality from this disease. The long non-coding RNA (lncRNA) SNHG1 is highly expressed in prostate cancer and is associated with poor progression-free survival. We investigated SNHG1 involvement in cell cycle regulation, quiescence, and chemoresistance. Suppression of SNHG1 expression using RNAi in PC3 and C4-2B prostate cancer cells resulted in a cessation of proliferation. SNHG1-deficient cells maintained viability, but did not increase in numbers over 7 days post-siRNA transfection. Control cells (siCTRL), meanwhile, expanded 12 and 10-fold, respectively. Analysis of DNA synthesis by EdU staining PC3 cells showed 32% EdU+ siCTRL, but only 7.0% EdU+ siSNHG1, cells. Likewise, C4-2B cells had reduced EdU staining after SNHG1 RNAi. We then analyzed cell cycle and apoptosis in C4-2B, Du145, and LNCaP cells by propidium iodide (PI) staining. Following SNHG1 RNAi, no significant increase in apoptosis or cell cycle arrest was observed, compared to controls. The reduction in S-phase cells was analogous to EdU staining, and there was a modest decrease in G2 phase cells. To investigate quiescence, we knocked down SNHG1 in PC3 cells engineered to express the fluorescent markers Venus and mCherry concurrently with expression of CDT1 and p27, as a marker of G0-phase cells. In SNHG1-deficient cells, 48% were in G0 phase (Venus+, mCherry+), while 13% of siCTRL cells were in G0 phase, indicating that loss of SNHG1 expression blocked cells from exiting G0 and entering the cell cycle. In C4-2B, knockdown of SNHG1 resulted in a 4-5-fold increase in p27(Kip1) expression by immunoblot, indicating increased numbers of quiescent cells. We assessed induction of apoptosis following docetaxel treatment in SNHG1-deficient cells. We found that 20 nM docetaxel induced a 2.9 and 11-fold increase in PARP cleavage in siCTRL-transfected PC3 and C4-2B cells compared to untreated, respectively. When SNHG1 was suppressed by RNAi, PARP cleavage following docetaxel treatment changed by 0.58 and 3.6-fold in PC3 and C4-2B cells, respectively. In addition, we found caspase 3 cleavage after docetaxel treatment was reduced following SNHG1 knockdown, compared to controls. These data indicate cells deficient in SNHG1 are more resistant to docetaxel. Because docetaxel also induced a G2 arrest, we quantified G2 phase by PI staining for DNA content, and immunoblotting for cyclin B1, a marker of G2/M, in siCTRL and siSNHG1 cells. There was a complete elimination of the docetaxel-induced G2 arrest in cells deficient in SNHG1. These data show that SNHG1 plays a role in prostate cancer cell cycle, which has an impact on response to chemotherapy. SNHG1-deficient cells accumulated in G0 phase, and there was reduced apoptosis and resistance to G2 arrest following docetaxel treatment. This is consistent with a model whereby low SNHG1 expression maintains dormancy and protects cells from treatment. Citation Format: Steven P. Zielske, Frank C. Cackowski. The lncRNA SNHG1 modulates quiescence and chemoresistance of prostate 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 1544.

Abstract 3643: The role of HBP1 in controlling breast cancer dormancy reawakening

Abstract At least 22% of breast cancer (BrCa) cases recur after 3 to 15 years, typically at metastatic sites such as the bone. Bone metastases derive from early disseminated tumor cells that become dormant because of interaction with specific cells that define the endosteal niche (EN) microenvironment. However, outside knowing that dormancy is driven by a p38MAPKhi, ERK1/2lo activation profile, our understanding of what factors regulate dormancy maintenance and reawakening is poor. We used a genomic shRNA screen to identify HBP1 as a suppressor of BrCa dormancy reawakening in a 3D-EN culture system. Our data indicate that under 3D-EN conditions, HBP1 is upregulated by p38MAPK, interacts with the metastasis suppressors, BRMS1 and BRMS1L, and with the histone modifiers, SIN3A and HDAC1-3. We hypothesize that HBP1 suppresses p38MAPK-mediated dormancy reawakening by complexing with BRMS1/BRMS1L/SIN3A to alter cell cycle gene expression through histone landscape remodeling. HBP1 suppresses the expression of cell cycle progression genes, such as MYC and CCND1, and concomitantly upregulates cyclin kinase inhibitors, p16 and p21. HBP1 RNA and protein levels are decreased in BrCa metastases relative to primary tumors, and this downregulation correlates with poorer overall survival. We are investigating whether the anti-histiminic drug, cyproheptadine, which upregulates HBP1 expression through the activation of p38MAPK, can suppress dormancy reawakening of BrCa cells in 3D-EN cultures and in in vivo models of bone dormancy using mouse mammary carcinoma D2A1 cell lines with varying abilities to form active or dormant metastatic bone colonization. Data will be shown addressing how cell-cell interactions and secretory factors between BrCa and bone EN cells facilitates dormancy signaling through a p38MAPK/HBP1/BRMS1 axis, and how drugs such as cyproheptadine agonize that pathway in order to prevent the reawakening that drives the growth of BrCa metastases in the bone, thereby increasing overall survival. Citation Format: Irwin H. Gelman, Seamus Degan, Henry G. Withers, Subrahmanya Anirudh Kaligotla Venkata. The role of HBP1 in controlling breast cancer dormancy reawakening [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 3643.

Stromal changes in the aged lung induce an emergence from melanoma dormancy.

Disseminated cancer cells from primary tumours can seed in distal tissues, but may take several years to form overt metastases, a phenomenon that is termed tumour dormancy. Despite its importance in metastasis and residual disease, few studies have been able to successfully characterize dormancy within melanoma. Here we show that the aged lung microenvironment facilitates a permissive niche for efficient outgrowth of dormant disseminated cancer cells-in contrast to the aged skin, in which age-related changes suppress melanoma growth but drive dissemination. These microenvironmental complexities can be explained by the phenotype switching model, which argues that melanoma cells switch between a proliferative cell state and a slower-cycling, invasive state1-3. It was previously shown that dermal fibroblasts promote phenotype switching in melanoma during ageing4-8. We now identify WNT5A as an activator of dormancy in melanoma disseminated cancer cells within the lung, which initially enables the efficient dissemination and seeding of melanoma cells in metastatic niches. Age-induced reprogramming of lung fibroblasts increases their secretion of the soluble WNT antagonist sFRP1, which inhibits WNT5Ain melanoma cells and thereby enables efficient metastatic outgrowth. We also identify the tyrosine kinase receptors AXL and MER as promoting a dormancy-to-reactivation axiswithin melanoma cells. Overall, we find that age-induced changes in distal metastatic microenvironments promote the efficient reactivation of dormant melanoma cells in the lung.