Dormant Cells Research Articles

Abstract A078: Response and resistance to KRAS inhibition in PDAC mouse models

Abstract Pancreatic ductal adenocarcinoma (PDAC), among the most lethal cancers with a 5-year survival rate of 11%, is a KRAS-driven disease. Studies have shown that a single glycine (G) to aspartic acid (D) substitution on codon 12 of KRAS protein (known as KRASG12D mutation) appears in 41% of all PDAC cases, highlighting the necessity of targeting KRASG12D as a therapeutic approach. MRTX1133, a recently developed potent, non-covalent, direct, and selective KRASG12D small molecule inhibitor demonstrates a high-affinity interaction with KRASG12D. Previous studies have shown striking efficacy of MRTX1133 on growth inhibition of mouse/human PDAC tumor cells carrying KRASG12D mutation in various in vivo and in vitro models, during short-term treatment. Here in our study, we investigated the efficacy of continuous long-term treatment with MRTX1133 in two different PDAC mouse models: KPC (a genetically engineered mouse model harboring a KRASG12D mutation) and its syngeneic orthotopic model. In both models, tumor sizes were detected by ultrasound imaging and after reaching 100-300 mm3 in volume, mice were enrolled in either MRTX1133 or vehicle treatment (30mg/kg, IP, BID). Tumor sizes were monitored by ultrasound regularly and the treatment continued until mice reached clinical endpoint. At endpoint, mice were sacrificed, and tumors were harvested for a series of downstream experiments. Our results showed that while, in both models, vehicle treated tumors grew aggressively and mice reached endpoint in average 6 days post treatment, drug treated tumors regressed rapidly and survival rate increased significantly to as late as 222 days post treatment in KPC model. However, in both models, drug treated tumors ultimately relapsed, developed resistance to the drug, and resulted in death. Further analysis of KPC mice ultrasound images evidenced that the relapsed tumors occurred at the same sites of the primary tumors. This suggests the possibility of relapsed tumors arising from the regrowth of the dormant residual cells from the primary tumors. Re-implantation of the resistant tumors either in the flank or pancreata of the new wildtype mice resulted in the formation of drug-resistant tumors in hosts. In addition, histopathology analysis of the tumor sections demonstrated a significant increase in the ratio of invasive PDAC to PanIN areas in the drug resistant tumors. Furthermore, multiplex immunofluorescence of both MRTX1133 resistant and vehicle treated tumors from KPC mice demonstrated co-expression of markers representing basal-like and classical PDAC subtypes. Moreover, flow cytometry analysis indicated an increase in the myeloid population of the resistant tumors comparing to the vehicle treated group. In the future directions, through applying a broad range of cellular and molecular experiments we will characterize the kinetics of KRAS inhibitor drug resistance and define possible combination therapy startegies to overcome drug resistance mechanisms. Citation Format: Laleh Abbassi, Jullien Dilly, Annan Yang, Giselle Uribe, Branden Parent, Connor Hennessey, Kevin Kapner, Alexander Jordan, Shatavisha Dasgupta, Micaela Morgado, Taimour Baslan, Li Qiang, Peter Winter, Seema Chugh, Alex Shalek, Ben Stanger, Srivastan Raghavan, Scott Lowe, Brian Wolpin, Stephanie Dougan, Jonathan Nowak, Andrew Aguirre. Response and resistance to KRAS inhibition in PDAC mouse models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A078.

Hematopoietic stem cell quiescence and DNA replication dynamics maintained by the resilient β-catenin/Hoxa9/Prmt1 axis

AbstractMaintenance of quiescence and DNA replication dynamics are 2 paradoxical requirements for the distinct states of dormant and active hematopoietic stem cells (HSCs), which are required to preserve the stem cell reservoir and replenish the blood cell system in response to hematopoietic stress, respectively. Here, we show that key self-renewal factors, β-catenin or Hoxa9, largely dispensable for HSC integrity, in fact, have dual functions in maintaining quiescence and enabling efficient DNA replication fork dynamics to preserve the functionality of hematopoietic stem and progenitor cells (HSPCs). Although β-catenin or Hoxa9 single knockout (KO) exhibited mostly normal hematopoiesis, their coinactivation led to severe hematopoietic defects stemmed from aberrant cell cycle, DNA replication, and damage in HSPCs. Mechanistically, β-catenin and Hoxa9 function in a compensatory manner to sustain key transcriptional programs that converge on the pivotal downstream target and epigenetic modifying enzyme, Prmt1, which protects the quiescent state and ensures an adequate supply of DNA replication and repair factors to maintain robust replication fork dynamics. Inactivation of Prmt1 phenocopied both cellular and molecular phenotypes of β-catenin/Hoxa9 combined KO, which at the same time could also be partially rescued by Prmt1 expression. The discovery of the highly resilient β-catenin/Hoxa9/Prmt1 axis in protecting both quiescence and DNA replication dynamics essential for HSCs at different key states provides not only novel mechanistic insights into their intricate regulation but also a potential tractable target for therapeutic intervention.

Identification of MCM4 and PRKDC as new regulators of osteosarcoma cell dormancy based on 3D cell cultures

Dormancy is a potential way for tumors to develop drug resistance and escape treatment. However, the mechanisms involved in cancer dormancy remain poorly understood. This is mainly because there is no in vitro culture model making it possible to spontaneously induce dormancy. In this context, the present work proposes the use of three-dimensional (3D) spheroids developed from osteosarcoma cell lines as a relevant model for studying cancer dormancy. MNNG-HOS, SaOS-2, 143B, MG-63, U2OS and SJSA-1 cell lines were cultured in 3D using the Liquid Overlay Technique (LOT). Dormancy was studied by staining cancer cells with a lipophilic dye (DiD), and long-term DiD+ cells were considered as dormant cancer cells. The role of the extracellular matrix in inducing dormancy was investigated by embedding cells into methylcellulose or Geltrex™. Gene expression of DiD+ cells was assessed with a Nanostring™ approach and the role of the genes detected in dormancy was validated by a transient down-expression model using siRNA treatment. Proliferation was measured using fluorescence microscopy and the xCELLigence technology. We observed that MNNG-HOS, 143B and MG-G3 cell lines had a reduced proliferation rate in 3D compared to 2D. U2OS cells had an increased proliferation rate when they were cultured in Geltrex™ compared to other 3D culture methods. Using 3D cultures, a transcriptomic signature of dormancy was obtained and showed a decreased expression of 18 genes including ETV4, HELLS, ITGA6, MCM4, PRKDC, RAD21 and UBE2T. The treatment with siRNA targeting these genes showed that cancer cell proliferation was reduced when the expression of ETV4 and MCM4 were decreased, whereas proliferation was increased when the expression of RAD21 was decreased. 3D culture facilitates the maintenance of dormant cancer cells characterized by a reduced proliferation and less differential gene expression as compared to proliferative cells. Further studies of the genes involved has enabled us to envisage their role in regulating cell proliferation.

The accumulation of methylated porphyrins in dormant cells of Mycolicibacterium smegmatis is accompanied by a decrease in membrane fluidity and an impede of the functioning of the respiratory chain

Transition of Mycolicibacterium smegmatis (Msm) and Mycobacterium tuberculosis to dormancy in vitro is accompanied by an accumulation of free methylated forms of porphyrins (tetramethyl coproporphyrin – TMC) localized in the cell wall of dormant bacteria. A study of the fluorescence anisotropy of BODIPY based fluorescent probes on individual cell level using confocal microscope revealed significant changes in this parameter for BODIPY FL C16 from 0.05 to 0.22 for vegetative and dormant Msm cells correspondingly. Similarly, the increase of TMC concentration in vegetative Msm cells grown in the presence of 5-aminolevulinic acid (a known inducer of porphyrin synthesis) resulted in an increase of BODIPY FL C16 anisotropy. These changes in TMC concentration and membrane fluidity were accompanied by an inhibition of the activity of the respiratory chain measured by oxygen consumption and a reduction of the DCPIP redox acceptor. During the first 8 h of the reactivation of the dormant Msm cells, the porphyrin content and probe fluorescent anisotropy returned to the level for vegetative bacteria. We suggested that upon transition to dormancy, an accumulation of TMC in membranes leads to a decrease in membrane fluidity, resulting in an inhibition of the respiratory chain activity. However, direct interactions of TMC with membrane bound enzymes cannot also be excluded. This, in turn, may result in the down regulation of many metabolic energy-dependent reactions as a part of mechanisms accompanying the transition to a hypometabolic state of mycobacteria.

MALAT1 protects dormant tumor cells from immune elimination.

MALAT1 protects dormant tumor cells from immune elimination.

The role of matrix metalloproteinase-2 in the metastatic cascade: a review

Abstract Matrix metalloproteinase-2 (MMP-2) is a gelatinase and is involved in multiple steps of the metastatic cascade. More than a decade ago an increased expression of MMP-2 in tumour cells or higher serum levels was reported to be a prognostic biomarker for a lower disease-free and overall survival rate. In recent years new evidence has indicated that MMP-2 has an important role in the tumour ecosystem. It is one of the many players in the onco-sphere, involved in interacting between tumour cells, host cells and the microenvironment. It plays a role in the dissemination of tumour cells, the epithelial–mesenchymal and mesenchymal–epithelial transitions, the formation of the pre-metastatic and metastatic niches, dormancy of tumour cells and modulating the immune system. The aim of this review is to highlight these multiple roles in the metastatic cascade and how many signalling pathways can up or down-regulate MMP-2 activity in the different stages of cancer progression and the effect of MMP-2 on the onco-sphere. Research in head and neck cancer is used as an example of these processes. The use of non-specific MMP inhibitors has been unsuccessful showing only limited benefits and associated with high toxicity as such that none have progressed past Phase III trials. Preclinical trials are undergoing using antibodies directed against specific matrix metalloproteinases, these targeted therapies may be potentially less toxic to the patients.

Unveiling the role of cellular dormancy in cancer progression and recurrence.

Cellular dormancy is a major contributor to cancer progression and recurrence. This review explores recent findings on the molecular mechanisms implicated in cancer dormancy and investigates potential strategies to improve therapeutic interventions. Research on cancer dormancy reveals a complex and multifaceted phenomenon. Providing a latent reservoir of tumor cells with reduced proliferation and enhanced drug-tolerance, dormant cancer cells emerge from a clonally diverse population after therapy or at metastatic sites. These cells exhibit distinct transcriptional and epigenetic profiles, involving the downregulation of Myc and mechanistic target of rapamycin (mTOR) pathways, and the induction of autophagy. Senescence traits, under the control of factors such as p53, also contribute significantly. The tumor microenvironment can either promote or prevent dormancy establishment, notably through the involvement of T and NK cells within the dormant tumor niche. Strategies to combat dormancy-related relapse include direct elimination of dormant tumor cells, sustaining dormancy to prolong survival, or awakening dormant cells to re-sensitize them to antiproliferative drugs. Improving our understanding of cancer dormancy at primary and secondary sites provides valuable insights into patient care and relapse prevention.

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.

Matrix stiffness influences response to chemo and targeted therapy in brain metastatic breast cancer cells.

Breast cancer is the most common malignancy accounting for 12.5% of all newly diagnosed cancer cases across the globe. Breast cancer cells are known to metastasize to distant organs (i.e., brain), wherein they can exhibit a dormant phenotype for extended time periods. These dormant cancer cells exhibit reduced proliferation and therapeutic resistance. However, the mechanisms by which dormant cancer cells exhibit resistance to therapy, in the context of brain metastatic breast cancer (BMBC), is not well understood. Herein, we utilized hyaluronic acid (HA) hydrogels with varying stiffnesses to study drug responsiveness in dormant vs. proliferative BMBC cells. It was found that cells cultured on soft HA hydrogels (∼0.4 kPa) that showed a non-proliferative (dormant) phenotype exhibited resistance to Paclitaxel or Lapatinib. In contrast, cells cultured on stiff HA hydrogels (∼4.5 kPa) that showed a proliferative phenotype exhibited responsiveness to Paclitaxel or Lapatinib. Moreover, dormancy-associated resistance was found to be due to upregulation of the serum/glucocorticoid regulated kinase 1 (SGK1) gene which was mediated, in part, by the p38 signaling pathway. Accordingly, SGK1 inhibition resulted in a dormant-to-proliferative switch and response to therapy. Overall, our study demonstrates that matrix stiffness influences dormancy-associated therapy response mediated, in part, via the p38/SGK1 axis.

Modeling Tumor Cell Dormancy in an Ex Vivo Liver Metastatic Niche.

Despite decades of research into metastatic disease, our knowledge of the mechanisms governing dormancy are still limited. Unraveling the process will aid in developing effective therapies to either maintain or eliminate these dormant cells and thus prevent them from emerging into overt metastatic disease. To study the behavior of dormant tumor cells-mechanisms that promote, maintain, and disrupt this state-we utilize the Legacy LiverChip®, an all-human ex vivo hepatic microphysiological system. This complex, bioengineered system is able to recreate metastatic disease that is reflective of the human situation and is among only a handful of systems able to mimic spontaneous tumor cell dormancy. The dormant subpopulation reflects the defining traits of cellular dormancy-survival in a foreign microenvironment, chemoresistance, and reversible growth arrest. This microphysiological system has and continues to provide critical insights into the biology of dormant tumor cells. It also serves as an accessible tool to identify new therapeutic strategies targeting dormancy and concurrently evaluate the efficacy of therapeutic agents as well as their metabolism and dose-limiting toxicity.

Isolating Cancer Stem Cells from Solid Tumors.

Over the past 20years, there has been a lot of interest in the study and investigation of cancer stem cells (CSCs) or tumor-initiating cells (TICs). CSCs are rare, dormant cells and able to self-renew and maintain tumor development and heterogeneity. A new age of basic and clinical cancer research, reclassification of human tumors, and the development of novel therapeutic approaches will undoubtedly result from a better knowledge of CSCs. In order to develop effective and therapeutic strategies to treat cancer, it is crucial to understand the basic characteristics of CSCs, their importance to cancer therapy, and methodologies to isolate, detect, and characterize them. Here, we outline the main methods and protocols to identify, isolate, and culture CSCs from primary tumors.

Detection of ROS in Dormant Breast Cancer Cell.

Reactive oxygen species (ROS) production can occur both as a physiological response and because of oxidative stress. ROS are not only the end product of nonfunctional cell processes but also signaling molecules that can regulate cell and tissue homeostasis. Recently, we have discovered that metastatic breast cancer cells that lay dormant in the lung microenvironment activate mitochondrial ROS production in response to the mechanical properties of the ECM, which triggers an antioxidant response mediated by the NRF2 transcription factor. In turn, this response protects dormant metastatic cells from cisplatin chemotherapy. Many tools have been developed to monitor ROS production in cells in culture, while our ability to detect this in vivo remains limited. Here we describe a detailed protocol for determination of ROS in metastatic cells in the mouse lung tissue by detecting 4-hydroxy-2-noneal (4HNE) adducts formation in fixed tissues.

Intracardiac Injection Mouse Model to Study Cancer Cell Dormancy in Brain Metastasis.

Brain metastasis is a highly complex process, and some cancer cells enter a dormant state after extravasation into the brain. The molecular mechanism of dormancy remains largely unknown and is still under intense investigation. Here, we outline a basic approach to generating and analyzing experimental mouse models to study dormant cancer cells in the brain. Cancer cells stably expressing EGFP and firefly luciferase are injected into the left ventricle of athymic nude mice. After confirmation of brain metastasis by bioluminescence imaging, brain slices are prepared and subjected to Ki67 staining. In addition, a methodology for recovering brain metastatic cancer cells from the mouse brain is described, providing technical tips for unraveling the mysteries of cancer cell dormancy in brain metastasis.

Immune evasion by dormant disseminated cancer cells: A Fermi paradox?

Rare disseminated tumor cells (DTCs) can persist after treatment in patients for years, and the immune system does not eliminate them. Goddard etal. propose that immune evasion by rare dormant DTCs is due to an improbability of contact imposed by large distances separating effector Tcells and DTCs.

Increased blood CSF3R+ myeloid-derived suppressor cell is a predictor for breast cancer recurrence.

Early detection of cancer recurrence using specific biomarkers remains a clinically unmet need, although methodologies for monitoring tumor markers, cell-free DNA, and circulating tumor cells have been established for decades. Tumor recurrence develops in metastatic or dormant cancer cells under continuous immune surveillance. Alterations in the population and function of immune cells may contribute to cancer recurrence. Here, we utilized an animal model to imitate breast tumor recurrence after surgical resection and investigated the abundance and gene expression profiles of immune cells using NanoString analysis. Bioinformatic analysis of a published single-cell RNA sequencing database of myeloid-derived suppressor cells (MDSCs) was performed to identify common targets between the two studies. Identified biomarkers were validated using human peripheral blood mononuclear cell (PBMC) datasets. The inhibitory effect of MDSCs on T-cell proliferation was assessed in vitro. Our data demonstrated that the number of MDSCs significantly increased during recurrence. Comparison of our NanoString data with a single-cell RNA sequencing dataset of MDSCs in another spontaneous breast cancer model identified colony-stimulating factor 3 receptor (Csf3r)-positive MDSCs as a potential marker for predicting tumor relapse. We validated our findings using two previously published PBMC databases of patients with breast cancer with or without recurrence and confirmed the elevated MDSC gene signature and CSF3R expression in patients with tumor recurrence. 35 patients with breast cancer were also included in our study, that patients with higher levels of CSF3R had worse survival. In vitro experiments demonstrated that Csf3r + MDSCs exhibited enhanced reactive oxygen species (ROS) levels and robust T-cell suppression ability. We conclude that an increase in CSF3R + MDSCs is a potential biomarker for early detection of tumor recurrence in patients with breast cancer.

Stress-Tolerant Dormant Bacterial Forms: Biological and Ultrastructural Properties of Moraxella catarrhalis and Kocuria rhizophila.

Dormant forms of causative agents of healthcare-acquired infections Moraxella catarrhalis and Kocuria rhizophila have been obtained. Dormant forms cells retained viability during long-term storage (≈107 CFU/ml after 2 months) under provocative conditions (lack of nutrient sources; temperature 20°C, oxygen access) were characterized by heat resistance, and acquired special ultrastructural organization typical of dormant forms (compacted nucleoid, thickened cell wall). They were also capable of forming alternative phenotypes (dominant and small colony variants) in a new cycle of germination in a fresh medium. These results demonstrate that the dormant forms can be responsible both for survival in the environment and persistence in the host organism.

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.

Neutrophil Extracellular Traps in Breast Cancer: Roles in Metastasis and Beyond.

Despite advances in the treatment of breast cancer, the disease continues to exhibit high global morbidity and mortality. The importance of neutrophils in cancer development has been increasingly recognized. Neutrophil extracellular traps (NETs) are web-like structures released into the extracellular space by activated neutrophils, serving as a potential antimicrobial mechanism for capturing and eliminating microorganisms. The roles played by NETs in cancer development have been a subject of intense research in the last decade. In breast cancer, current evidence suggests that NETs are involved in various stages of cancer development, particularly during metastasis. In this review, we try to provide an updated overview of the roles played by NETs in breast cancer metastasis. These include: 1) facilitating systemic dissemination of cancer cells; 2) promoting cancer-associated inflammation; 3) facilitating cancer-associated thrombosis; 4) facilitating pre-metastatic niche formation; and 5) awakening dormant cancer cells. The translational implications of NETs in breast cancer treatment are also discussed. Understanding the relationship between NETs and breast cancer metastasis is expected to provide important insights for developing new therapeutic strategies for breast cancer patients.

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.

Cancer Stem Cells: Detection and Characterization from Solid Tumors.

Cancer stem cells (CSCs) have emerged as an attractive research interest due to their prominent role in development of the tumors. CSCs are rare dormant cells that can self-renew and maintain tumor development and heterogeneity. A better understanding of CSCs can improve tumor classification and contribute toward the development of novel therapeutic approaches to fight cancer. Hence, it is of immense importance to comprehend the basic function of CSCs in tumor formation, which can only be possible by devising perfected methodologies to isolate, detect, and characterize them. In this chapter, we outline the key protocols to culture, identify, and isolate CSCs from solid tumors to further advance basic and clinical investigation related to CSCs and their role in tumor biology.