Breast Cancer Dormancy Research Articles

Mitochondrial A10398G Alteration in Plasma Exosome of Non-small Cell Lung Cancer Patients

This study identifies A10398G alteration of mitochondrial ND3 gene in plasma exosome of 29 non-small cell lung cancer (NSCLC) patients, 31 controls and 13 pairs of tumor tissue and adjacent tissue of NSCLC patients, thereby assessing the relationship between this alteration in plasma exosome and tissue as well as the pathological characteristics of NSCLC patients. Using the PCR-RFLP method, the homoplasmy and heteroplasmy of A10398G were initially identified in mitochondrial DNA from both exosomes and lung tissues. The rate of variant 10398G in plasma exosome was 62.1% in the NSCLC group and 61.3% in the control group. However, there was no statistically significant difference in A10398G between the patient and control groups. The alteration of A10398G in plasma exosome and in tissue correlated with each other (correlation coefficient 0.69; p = 0.009). However, this alteration was not related to age, gender, smoking, alcohol drinks status, tumor size, histological stage and TNM stage.
 Keywords
 A10398G alteration, mitochondrial DNA, plasma exosome, non-small cell lung cancer.
 References
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Specific N-Cadherin-Dependent Pathways Drive Human Breast Cancer Dormancy in Bone Marrow

The challenge for treating breast cancer (BC) is partly due to long-term dormancy driven by formation of cancer stem cells (CSCs) capable of evading immune response and resist chemotherapy. BC cells (BCCs) show preference for bone marrow (BM), resulting in poor prognosis. CSCs utilize connexin 43 (Cx43) to form gap junctional intercellular communication (GJIC) with BM niche cells, fibroblasts and mesenchymal stem cells (MSCs). However, Cx43 is an unlikely target to reverse BC dormancy due to its role as hematopoietic regulator. We found N-cadherin (CDH2) and its associated pathways as potential drug targets. CDH2, highly expressed in CSCs, interacts intracellularly with Cx43, co-localizes with Cx43 in BCCs within BM biopsies of patients, and is required for Cx43-mediated GJIC with BM niche cells. Notably, CDH2-associated γ-secretase and pro-apoptotic pathways maintained BC dormancy. We thereby propose these pathways as potential pharmacological targets to prevent dormancy and chemosensitize resistant CSCs.

Breast Cancer Dormancy: Understanding ER+ Breast Cancer Dormancy Using Bioinspired Synthetic Matrices for Long‐Term 3D Culture and Insights into Late Recurrence (Adv. Biosys. 9/2020)

In article number 2000119, April M. Kloxin and co-workers establish a 3D human breast cancer dormancy model that captures key biophysical and biochemical properties of common metastatic microenvironments for late recurrence. The studies with this model demonstrate how matrix density and cell type impact cell dormancy versus proliferation and suggests autophagy as a survival mechanism of dormant ER+ breast cancer cells.

Abstract 2646: CD39+PD-1+CD8+ T cells mediate metastatic dormancy in breast cancer

Abstract Although metastatic disease can be present already at diagnosis, in some patients it appears months or years after resection of the primary tumor. This is thought to be due to the ability of some tumor cells to remain dormant after metastatic seeding. It is not known how dormancy is controlled, but there is evidence for tumor cell-intrinsic and -extrinsic mechanisms including immune defense. Here we found in preclinical models of breast cancer that metastatic dormancy was fully controlled by T cells. Through high-parametric single-cell mapping, we identified a discrete population of CD39+PD-1+CD8+ T cells present both in primary tumors and in spontaneous dormant metastasis, whereas this population is hardly found in aggressively metastasizing tumors. Surprisingly, despite expressing many exhaustion markers, this population was rich in effector molecules and cytokines that induced dormancy in tumor cells. Of note, the adoptive transfer of purified CD39+PD-1+CD8+ T cells prevented metastatic outgrowth. Thus, we discovered that primary breast tumors prime CD39+PD-1+CD8+ T cells that induce metastatic dormancy in distal disseminated tumor cells in the lungs. Citation Format: Paulino Tallón de Lara, Héctor Castañón Cuadrado, Marijne Vermeer, Nicolás Núñez, Joaquín Urdínez, Virginia Cecconi, Farkhondeh Movahedian Attar, Isabelle Glarner, Stefanie Hiltbrunner, Sonia Tugues, Burkhard Becher, Maries van den Broek. CD39+PD-1+CD8+ T cells mediate metastatic dormancy in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2646.

The Influence of Matrix-Induced Dormancy on Metastatic Breast Cancer Chemoresistance.

Metastasis remains the leading cause of cancer-associated death worldwide. Disseminated tumor cells can undergo dormancy upon infiltration of secondary organs, and chemotherapeutics fail to effectively eliminate dormant populations. Mechanistic understanding of dormancy-associated chemoresistance could lead to development of targeted therapeutic strategies. Toward this goal, we implemented three poly(ethylene glycol) (PEG)-based hydrogel formulations fabricated from proteolytically degradable PEG (PEG-PQ), integrin ligating PEG-RGDS, and the non-degradable cross-linker N-vinylpyrrolidone (NVP) to induce three distinct phenotypes in triple negative MDA-MB-231 breast cancer cells. With constant 5% w/v PEG-PQ, PEG-RGDS and NVP concentrations were tuned to induce (i) a growth state characterized by high proliferation, high metabolic activity, significant temporally increased cell density, and an invasive morphology; (ii) a balanced dormancy state characterized by a temporal balance (~1:1 ratio) in new live and dead cell density and a non-invasive morphology; and (iii) a cellular dormancy state characterized by rounded, solitary quiescent cells with low viability, proliferation, and metabolic activity. The cellular responses to doxorubicin (DOX), paclitaxel (PAC), and 5-fluorouracil (5-FU) in the three phenotypic states were quantified. Under DOX treatment, cells in dormant states demonstrated increased chemoresistance with a 1.4- to 1.8-fold increase in half maximal effective concentration (EC50) and 1.3- to 1.8-fold increase in half maximal inhibitory concentration (IC50) compared to cells in the growth state. PAC and 5-FU treatment led to similar results. To mechanistically investigate the role of dormancy in conferring DOX resistance, cytoplasmic and nuclear accumulation of DOX was measured. The results indicated comparable DOX accumulation between all three phenotypic states; however, the intracellular to intranuclear distribution indicated a ~1.5 fold increase in DOX nuclear accumulation in cells in the growth state compared to the two dormant states. These results further validate the utility of implementing engineered hydrogels as in vitro platforms of breast cancer dormancy for the development of anti-dormancy therapeutic strategies.

Understanding ER+ Breast Cancer Dormancy Using Bioinspired Synthetic Matrices for Long-Term 3D Culture and Insights into Late Recurrence.

Late recurrences of breast cancer are hypothesized to originate from disseminated tumor cells that re-activate after a long period of dormancy, ≥5 years for estrogen-receptor positive (ER+) tumors. An outstanding question remains as to what the key microenvironment interactions are that regulate this complex process, and well-defined human model systems are needed for probing this. Here, a robust, bioinspired 3D ER+ dormancy culture model is established and utilized to probe the effects of matrix properties for common sites of late recurrence on breast cancer cell dormancy. Formation of dormant micrometastases over several weeks is examined for ER+ cells (T47D, BT474), where the timing of entry into dormancy versus persistent growth depends on matrix composition and cell type. In contrast, triple negative cells (MDA-MB-231), associated with early recurrence, are not observed to undergo long-term dormancy. Bioinformatic analyses quantitatively support an increased "dormancy score" gene signature for ER+ cells (T47D) and reveal differential expression of genes associated with different biological processes based on matrix composition. Further, these analyses support a link between dormancy and autophagy, a potential survival mechanism. This robust model system will allow systematic investigations of other cell-microenvironment interactions in dormancy and evaluation of therapeutics for preventing late recurrence.

Hypoxia-mediated changes in bone marrow microenvironment in breast cancer dormancy.

Breast cancer (BC) remains a clinical challenge despite improved treatments and public awareness to ensure early diagnosis. A major issue is the ability of BC cells (BCCs) to survive as dormant cancer cells in the bone marrow (BM), resulting in the cancer surviving for decades with the potential to resurge as metastatic cancer. The experimental evidence indicates similarity between dormant BCCs and other stem cells, resulting in the preponderance of data to show dormant BCCs being cancer stem cells (CSCs). The BM niche and their secretome support BCC dormancy. Lacking in the literature is a comprehensive research to describe how the hypoxic environment within the BM may influence the behavior of BCCs. This information is relevant to understand the prognosis of BC in young and aged individuals whose oxygen levels differ in BM. This review discusses the changing information on vascularity in different regions of the BM and the impact on endogenous hematopoietic stem cells (HSCs). This review highlights the necessary information to provide insights on vascularity of different BM regions on the behavior of BCCs, in particular a dormant phase. For instance, how the transcription factor HIF1-α (hypoxia-inducible factor 1 alpha), functioning as first responder under hypoxic conditions, affects the expression of specific gene networks involved in energy metabolism, cell survival, tumor invasion and angiogenesis. This enables cell fate transition and facilitates tumor heterogeneity, which in turn favors tumor progression and resistance to anticancer treatments Thus, HIF1-α could be a potential target for cancer treatment. This review describes epigenetic mechanisms involved in hypoxic responses during cancer dormancy in the bone marrow. The varied hypoxic environment in the BM is relevant to understand the complex process of the aging bone marrow for insights on breast cancer outcome between the young and aged.

Abstract B44: The role of MDSCs in breast cancer dormancy and recurrence

Abstract Tumor relapse and metastasis are a major cause of death in women with breast cancer. By the time of primary tumor diagnosis cancer has already metastasized, and patients have developed either metastatic tumors or dormant disseminated tumor cells (DTCs). Tumor dormancy is a specific stage in which residual cells remain inactive and therapy resistant, possibly due to quiescence, niche protection, or both. To understand the obstacles to overcome protumorigenic signaling stemming from microenvironment, we are utilizing various syngeneic model of residual disease/dormancy and recurrence. We found that myeloid-derived suppressor cells (MDSCs) are recruited to tumor following the FGFR-mTOR pathway activation and enhance tumor-initiating cells through direct contact by promoting Notch signaling in vitro. We have utilized a novel transgenic model, MMTV-WNT1-iFGFR1GFP (iFGFR1GFP), with an intact immune system to study residual disease, dormancy, and their relapse. We have generated primary tumors by transplanting and expanding spontaneous iFGFR1GFP tumors into fat pad of the glow-head mice. To induce dormancy, we have treated established tumors with BGJ398, a specific FGFR inhibitor for two weeks, a time at which they were barely palpable, followed by a further two weeks of recovery. Inhibition of FGFR results in residual disease, and cells remain in dormant stage for up to 4 months post-BGJ398 treatment. Additionally, the expression of GFP and luciferase reporters will facilitate both the localization and isolation of solitary DTCs at the metastatic sites (lung). Our results show that majority of GFP+ residual cells are Ki67 negative and form normal-like gland structures and there is a significant reorganization of tumor stroma. Our preliminary data suggest that Notch signaling is activated during recurrence. MDSCs are present at all stages, and their abundance increases as cells escape dormancy and relapse, suggesting that MDSCs may potentially promote Notch signaling and enhance recurrence. Single-cell sequencing of tumors at different stages has shown that MDSCs express Jagged-1 and are being recruited to tumor during the dormant stage. Blocking antibodies against Jagged-1 are being utilized to test if suppressing MDSCs will inhibit tumor recurrence. Our results show that anti-Jagged-1 antibody increases the time to relapse. Furthermore, we will perform tail vein injection of cells to investigate dormancy in lung, as well as resect primary tumors to enhance potential metastatic lesions’ growth. The impact of this research will be to provide potential early biomarkers to detect the presence of MDSCs in order to predict which patients might relapse as well as the discovery of combinatorial therapeutics to decrease tumor recurrence. Citation Format: Mahnaz Janghorban, Xiang Zhang, Jeffrey Rosen. The role of MDSCs in breast cancer dormancy and recurrence [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B44.

Breast Cancer Dormancy in Bone.

The goal of this review is to summarize recent experimental and clinical evidence for metastatic latency and the molecular mechanisms that regulate tumor dormancy in the bone. Tumor dormancy contributes to the progression of metastasis and thus has significant clinical implications for prognosis and treatment. Tumor-intrinsic signaling and specialized bone marrow niches play a pivotal role in determining the dormancy status of bone disseminated tumor cells. Experimental models have provided significant insight into the effects of the bone microenvironment on tumor cells; however, these models remain limited in their ability to study dormancy. Despite recent advances in the mechanistic understanding of how tumor cells remain dormant in the bone for prolonged periods of time, the signals that trigger spontaneous dormancy escape remain unclear. This review highlights the need for further investigation of mechanisms underlying tumor dormancy using clinically relevant models.

Chemotherapy-induced immunological breast cancer dormancy: a new function for old drugs?

The Journal of Cancer Metastasis and Treatment is an open access journal focused on cancer metastasis and treatment, including the occurrence, development, progression, metastasis, and treatment of oncologic disease. It covers basic, translational and clinical research related to cancer cell biology, genomics, precision medicine, oncology internal medicine, radiotherapy and radiology, obstetrics and gynecology, pediatrics, surgery, hematology, neurooncology, etc.

Tunable hydrogels for controlling phenotypic cancer cell states to model breast cancer dormancy and reactivation

During metastasis, disseminated tumor cells (DTCs) from the primary tumor infiltrate secondary organs and reside there for varying lengths of time prior to forming new tumors. The time delay between infiltration and active proliferation, known as dormancy, mediates the length of the latency period. DTCs may undergo one of four fates post-infiltration: death, cellular dormancy, dormant micrometastasis, or invasive growth which, is in part, mediated by extracellular matrix (ECM) properties. Recapitulation of these cell states using engineered hydrogels could facilitate the systematic and controlled investigation of the mechanisms by which ECM properties influence DTC fate. Toward this goal, we implemented a set of sixteen hydrogels with systematic variations in chemical (ligand (RGDS) density and enzymatic degradability) and mechanical (elasticity, swelling, mesh size) properties to investigate their influence on the fate of encapsulated metastatic breast cancer cells, MDA-MB-231. Cell viability, apoptosis, proliferation, metabolic activity, and morphological measurements were acquired at five-day intervals over fifteen days in culture. Analysis of the phenotypic metrics indicated the presence of four different cell states that were classified as: (1) high growth, (2) moderate growth, (3) single cell, restricted survival, dormancy, or (4) balanced dormancy. Correlating hydrogel properties with the resultant cancer cell state indicated that ligand (RGDS) density and enzymatic degradability likely had the most influence on cell fate. Furthermore, we demonstrate the ability to reactivate cells from the single cell, dormant state to the high growth state through a dynamic increase in ligand (RGDS) density after forty days in culture. This tunable engineered hydrogel platform offers insight into matrix properties regulating tumor dormancy, and the dormancy-proliferation switch, and may provide future translational benefits toward development of anti-dormancy therapeutic strategies.

Identification of Genes Regulating Breast Cancer Dormancy in 3D Bone Endosteal Niche Cultures.

Tumor cell dormancy is a significant clinical problem in breast cancer. We used a three-dimensional (3D) in vitro model of the endosteal bone niche (EN), consisting of endothelial, bone marrow stromal cells, and fetal osteoblasts in a 3D collagen matrix (GELFOAM), to identify genes required for dormancy. Human triple-negative MDA-MB-231 breast cancer cells, but not the bone-tropic metastatic variant, BoM1833, established dormancy in 3D-EN cultures in a p38-MAPK-dependent manner, whereas both cell types proliferated on two-dimensional (2D) plastic or in 3D collagen alone. "Dormancy-reactivation suppressor genes" (DRSG) were identified using a genomic short hairpin RNA (shRNA) screen in MDA-MB-231 cells for gene knockdowns that induced proliferation in the 3D-EN. DRSG candidates enriched for genes controlling stem cell biology, neurogenesis, MYC targets, ribosomal structure, and translational control. Several potential DRSG were confirmed using independent shRNAs, including BHLHE41, HBP1, and WNT3. Overexpression of the WNT3/a antagonists secreted frizzled-related protein 2 or 4 (SFRP2/4) and induced MDA-MB-231 proliferation in the EN. In contrast, overexpression of SFRP3, known not to antagonize WNT3/a, did not induce proliferation. Decreased WNT3 or BHLHE41 expression was found in clinical breast cancer metastases compared with primary-site lesions, and the loss of WNT3 or BHLHE41 or gain of SFRP1, 2, and 4 in the context of TP53 loss/mutation correlated with decreased progression-free and overall survival. IMPLICATIONS: These data describe several novel, potentially targetable pathways controlling breast cancer dormancy in the EN.

Prevention of cancer dormancy by Fbxw7 ablation eradicates disseminated tumor cells.

Dormant cancer cells known as disseminated tumor cells (DTCs) are often present in bone marrow of breast cancer patients. These DTCs are thought to be responsible for the incurable recurrence of breast cancer. The mechanism underlying the long-term maintenance of DTCs remains unclear, however. Here, we show that Fbxw7 is essential for the maintenance of breast cancer dormancy. Genetic ablation of Fbxw7 in breast cancer cells disrupted the quiescence of DTCs, rendering them proliferative, in mouse xenograft and allograft models. Fbxw7-deficient DTCs were significantly depleted by treatment with paclitaxel, suggesting that cell proliferation induced by Fbxw7 ablation sensitized DTCs to chemotherapy. The combination of Fbxw7 ablation and chemotherapy reduced the number of DTCs even when applied after tumor cell dissemination. Mice injected with Fbxw7-deficient cancer cells survived longer after tumor resection and subsequent chemotherapy than did those injected with wild-type cells. Furthermore, database analysis revealed that breast cancer patients whose tumors expressed FBXW7 at a high level had a poorer prognosis than did those with a low FBXW7 expression level. Our results suggest that a wake-up strategy for DTCs based on Fbxw7 inhibition might be of value in combination with conventional chemotherapy for the treatment of breast cancer.

Abstract P5-04-03: Molecular characterisation of ER+ breast cancer dormancy and acquired resistance using a clinical model: Potential involvement of epigenetic regulation

Abstract Background: The risk of recurrence for oestrogen receptor positive (ER+) breast cancer patients treated with 5 years of adjuvant endocrine therapy persists for many years or even decades following surgery and apparently successful adjuvant therapy. This period of dormancy and acquired resistance is inherently difficult to investigate. Therefore, previous efforts have been limited to in vitro or in vivo approaches. In this study sequential tumour samples from patients receiving extended neoadjuvant endocrine treatment were characterised as a novel clinical model of ER+ breast cancer dormancy and acquired resistance. Methods: Consecutive tumour samples from 62 patients undergoing extended (4-45 months) neoadjuvant letrozole therapy were subjected to transcriptomic and proteomic analysis, representing pre- (before treatment), early-on (13-120 days) and long-term (>120 days) neoadjuvant letrozole treatment. Patients with at least a 40% initial reduction in tumour size by 4 months of treatment were included. Of these, 42 patients with no subsequent progression were classified as “dormant”, and the remaining 20 patients as “acquired resistant”. Expression analysis was performed by using Illumina BeadChips. R and BioConductor packages were used for analysis. Differentially expressed genes were determined by using paired Rank Products (FDR, 5%). Results: Multidimensional scaling using most variant 500 genes demonstrated that long-term treated dormant samples clustered separately from their matched pre- and early-on samples whereas long-term treated resistant samples were indistinguishable from their pre-treatment counterparts. Therapy-induced changes in resistant tumours were common features of treatment, rather than being specific to resistant phenotype. Comparative analysis of long-term treated dormant and resistant tumours highlighted changes in epigenetics pathways including DNA methylation and histone acetylation. DNA methylation marks 5-methylcytosine and 5-hydroxymethylcytosine were significantly reduced in resistant tumours compared to dormant tissues after extended letrozole treatment. Decrease in 5-hydroxymethylcytosine were significant early-on. Conclusions: This is the first patient-matched gene expression study investigating long-term aromatase inhibitor-induced dormancy and acquired resistance in breast cancer. Dormant tumors exhibit distinct molecular changes under extended treatment whereas acquired resistant tumors are more similar to matched diagnostic samples supporting the molecular concordance between primary tumors and metastases. Global loss of DNA methylation was observed in resistant tumours under extended treatment which can be predicted within first 4 months of therapy. Epigenetic alterations may lead to escape from dormancy and drive acquired resistance in a subset of patients supporting a potential role for therapy targeted at these epigenetic alterations in the management of endocrine resistant breast cancer. Funding: This work was supported by Marie Skłodowska-Curie Individual Fellowship [H2020-MSCA-IF, 658170] and Welcome Trust Institutional Fund (ISSF3) to CS and AHS, Breast Cancer Now to AHS. Citation Format: Selli C, Turnbull AK, Pearce D, Fernando A, Renshaw L, Thomas JS, Dixon MJ, Sims AH. Molecular characterisation of ER+ breast cancer dormancy and acquired resistance using a clinical model: Potential involvement of epigenetic regulation [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-03.

Abstract P5-07-06: Investigating breast cancer dormancy in response to microenvironment cues with well-defined synthetic extracellular matrices

Abstract Breast cancer recurs in approximately 20% of disease-free patients 5+ years after successful treatment of the original tumor. These late recurrences often occur at metastatic sites, including the bone marrow and lungs, and are hypothesized to arise from disseminated tumor cells (DTCs) that reactivate after a long period dormancy. Interactions between DTCs and the microenvironment at these sites are thought to be important regulators of both breast cancer cell dormancy and reactivation. New approaches are needed to study this complex process and test hypotheses about key cell-microenvironment interactions in dormancy toward improved treatment strategies. In this work, we investigated the role of cell-matrix and cell-cell interactions on dormancy and activation of breast cancer cells utilizing well-defined materials to mimic the extracellular matrix (ECM) of metastatic sites and selectively introducing metastatic niche cells via dynamic co-culture. We established synthetic ECMs with well-defined properties that mimicked key aspects of the mechanical properties and biochemical content of bone marrow and lung metastatic sites (Young's modulus E˜0.5-5 kPa, respectively, and rich in collagen). These synthetic ECMs were constructed with a bioinert, multifunctional poly(ethylene glycol) crosslinked with a cell-degradable peptide and decorated with integrin-binding peptides derived from collagen and laminin, creating three-dimensional (3D) environments for the culture of breast cancer cells and niche cells. We cultured breast cancer cells of different metastatic potential (estrogen receptor positive [ER+, T47Ds] and triple negative [ER-, MDA-MB-231s]) within matrices, with or without co-culture of niche cells, and examined differences in markers of dormancy for up to 6 weeks. Both breast cancer cell types exhibited good viability in 3D culture (> 90%). Different degrees of cell proliferation (metabolic activity, EdU assay, cell/cluster number and volume) were observed over the first 2 weeks in culture, dependent upon matrix composition. Interestingly, ER- MDA-MB-231s were more responsive to differences in the biochemical content of the matrix, with increased elongation and proliferation observed within collagen mimic environments, whereas fewer differences were observed in the responses of the ER+ T47Ds to these same compositions. Further, both cell types exhibited significant decreases in proliferation in response to increased matrix density, mimicking the moduli of bone marrow to lung tissues. Dormant single cells or small cell clusters, reminiscent of micrometastases, were observed over weeks 2-6. Immunostaining and bioinformatics analyses revealed increased expression of autophagy markers for these dormant cells, suggesting a potential mechanism of survival. Additionally, co-culture with specific niche cells, including human mesenchymal stem cells and osteoblasts, was observed to enhance or suppress breast cancer cell proliferation within these well-defined environments. These studies have established a new controlled 3D breast cancer dormancy culture model, with on-going investigations examining cell re-activation for the identification of therapeutics to maintain dormancy and prevent recurrence. Citation Format: Kloxin AM, Ovadia EM, Pradhan L, Sawicki LA. Investigating breast cancer dormancy in response to microenvironment cues with well-defined synthetic extracellular matrices [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-07-06.

Abstract ES5-1: Tumor dormancy and recurrence

Abstract Despite early detection and adjuvant therapy, breast cancer remains the leading cause of cancer mortality in women, largely due to distant, incurable recurrences that arise years, or even decades, after treatment of the primary tumor. Recurrent, metastatic tumors arise from the pool of residual local and disseminated tumor cells (DTCs) that survive primary treatment in tissues at sites throughout the body where they persist in a presumed dormant state. Early studies demonstrated that an immunohistochemical assay can detect cytokeratin-positive DTCs in bone marrow aspirates of a substantial fraction of breast cancer patients lacking any clinical signs of metastasis. Importantly, women who harbor detectable DTCs in their bone marrow have a substantially increased risk of distant recurrence, as well as poorer breast cancer-specific and overall survival, and this measure has repeatedly been shown to be an independent predictor of recurrence-free survival and overall survival in multivariate analysis. Since DTCs constitute the reservoir from which recurrent cancers arise, understanding their biology is a critical priority in cancer research. We hypothesize that effectively disabling the survival mechanisms by which DTCs persist in breast cancer patients following treatment will deplete this critical reservoir of cells, reduce tumor recurrence, and thereby improve survival. At present, however, the mechanisms enabling these cells to survive in a dormant state and ultimately recur are poorly understood and clinical methods to detect DTCs, as well as DTC-directed therapeutic approaches, are still in their infancy. To address this critical gap and elucidate critical pathways in dormant tumor cell survival, we and others have developed a variety of mouse models for human breast cancer that recapitulate key features of human breast cancer progression, including metastasis, residual disease, dormancy and recurrence. Investigation of these models using both genetic and pharmacological approaches has identified several mediators of dormant DTC survival and recurrence, demonstrated their relevance to pathways that contribute to therapeutic resistance in human breast cancers following targeted therapy or chemotherapy, and revealed that pharmacological targets for dormancy and recurrence may be unique to these stages of tumor progression. These findings have, in turn, led to the initiation of clinical trials to test the ability of targeted therapies to deplete the burden of DTCs in the bone marrow of breast cancer patients and thereby decrease risk of relapse. If successful, the ability to therapeutically target survival mechanisms utilized by DTCs would constitute a powerful, paradigm-shifting approach to preventing cancer recurrence and the mortality associated with it. Citation Format: Chodosh L. Tumor dormancy and recurrence [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr ES5-1.

Exosomes from differentially activated macrophages influence dormancy or resurgence of breast cancer cells within bone marrow stroma

Breast cancer (BC) cells (BCCs) can retain cellular quiescence for decades, a phenomenon referred to as dormancy. BCCs show preference for the bone marrow (BM) where they can remain dormant for decades. Targeting BCCs within the BM is a challenge since the dormant BCCs reside within BM stroma, also residence for hematopoietic stem cells (HSCs). Dormant BCCs could behave as cancer stem cells (CSCs). The CSCs and HSCs are similar by function and also, by commonly expressed genes. The method by which dormant BCCs transition into clinically metastatic cells remains unclear. This study tested the hypothesis that macrophages (MΦs) within BM stroma, facilitates dormancy or reverse this state into metastatic cells. MΦs exhibiting an M2 phenotype constitute ~10% of cultured BM stroma. The M2 MΦs form gap junctional intercellular communication (GJIC) with CSCs, resulting in cycling quiescence, reduced proliferation and carboplatin resistance. In contrast, MΦs expressing the M1 phenotype reversed BC dormancy. Activation of M2a MΦs via the toll-like receptor 4 (TLR4) switched to M1 phenotype. The switch can occur by direct activation of M2a MΦs, or indirectly through activation of mesenchymal stem cells. M1 MΦ-derived exosomes activated NFкB to reverse quiescent BCCs to cycling cells. Using an in vivo model of BC dormancy, injected Mi MOs sensitized BCCs to carboplatin and increased host survival. In summary, we have shown how BM stromal MΦs, through exosomes, regulate the behavior of BCCs, by either inducing or reversing dormancy.

Fabrication, characterization, and implementation of engineered hydrogels for controlling breast cancer cell phenotype and dormancy

A better understanding of how microenvironmental factors regulate cancer dormancy is needed for development of new therapeutic strategies to control metastatic recurrence and disease progression. Modeling cancer dormancy using engineered, in vitro platforms is necessary for investigation under well-defined and well-controlled microenvironments. We present methods and protocols to fabricate, characterize, and implement engineered hydrogels with well-defined biochemical and physical properties for control over breast cancer cell phenotype in three-dimensional (3D) culture. Changes in hydrogel adhesivity, crosslink density, and degradability induce a range of phenotypic behaviors in breast cancer cells including: (1) high growth, (2) moderate growth, (3) single cell, restricted survival dormancy, and (4) balanced dormancy. We describe a method of classifying hydrogel formulations that support each of these phenotypic states. We also describe a method to phenotypically switch cancer cells from single cell dormancy to high growth by dynamically modulating ligand density, thereby recapitulating reactivation and metastatic recurrence.

Neoadjuvant Therapy for Breast Cancer as a Model for Translational Research.

Neoadjuvant therapy, where patients receive systemic therapy before surgical removal of the tumour, can downstage tumours allowing breast-conserving surgery, rather than mastectomy. In addition to its impact on surgery, the neoadjuvant setting offers a valuable opportunity to monitor individual tumour response. The effectiveness of standard and/or potential new therapies can be tested in the neoadjuvant pre-surgical setting. It can potentially help to identify markers differentiating patients that will potentially benefit from continuing with the same or a different adjuvant treatment enabling personalised treatment. Characterising the molecular response to treatment over time can more accurately identify the significant differences between baseline samples that would not be identified without post-treatment samples. In this review, we discuss the potential and challenges of using the neoadjuvant setting in translational breast cancer research, considering the implications for improving our understanding of response to treatment, predicting therapy benefit, modelling breast cancer dormancy, and the development of drug resistance.

Type I interferon/IRF7 axis instigates chemotherapy-induced immunological dormancy in breast cancer

Neoadjuvant and adjuvant chemotherapies provide survival benefits to breast cancer patients, in particular in estrogen receptor negative (ER−) cancers, by reducing rates of recurrences. It is assumed that the benefits of (neo)adjuvant chemotherapy are due to the killing of disseminated, residual cancer cells, however, there is no formal evidence for it. Here, we provide experimental evidence that ER− breast cancer cells that survived high-dose Doxorubicin and Methotrexate based chemotherapies elicit a state of immunological dormancy. Hallmark of this dormant phenotype is the sustained activation of the IRF7/IFN-β/IFNAR axis subsisting beyond chemotherapy treatment. Upregulation of IRF7 in treated cancer cells promoted resistance to chemotherapy, reduced cell growth and induced switching of the response from a myeloid derived suppressor cell-dominated immune response to a CD4+/CD8+ T cell-dependent anti-tumor response. IRF7 silencing in tumor cells or systemic blocking of IFNAR reversed the state of dormancy, while spontaneous escape from dormancy was associated with loss of IFN-β production. Presence of IFN-β in the circulation of ER− breast cancer patients treated with neoadjuvant Epirubicin chemotherapy correlated with a significantly longer distant metastasis-free survival. These findings establish chemotherapy-induced immunological dormancy in ER− breast cancer as a novel concept for (neo)adjuvant chemotherapy activity, and implicate sustained activation of the IRF7/IFN-β/IFNAR pathway in this effect. Further, IFN-β emerges as a potential predictive biomarker and therapeutic molecule to improve outcome of ER− breast cancer patients treated with (neo)adjuvant chemotherapy.