Disseminated tumour cells in bone marrow are the source of cancer relapse after therapy

Abstract Accumulating evidence indicates that cancer cells spread much earlier than was previously believed. Recent technological advances have greatly improved the detection methods of circulating tumour cells (CTCs), suggesting that the dissemination of cancer cells into the circulation occurs randomly. Most CTCs die in circulation as a result of shear stress and/or anoikis. However, the persistence of disseminated tumour cells (DTCs) in the bone marrow is the result of interaction of DTCs with bone marrow microenvironment. DTCs in the bone marrow undergo successive clonal expansions and a parallel progression that leads to new variants. Compared to the CTCs, DTCs in the bone marrow have a unique signature, which displayed dormant, mesenchymal phenotype and osteoblast‐like or osteoclast‐like phenotype. The persistence of DTCs in the bone marrow is always related to minimal residual diseases (MRDs). This review outlines the difference between CTCs and DTCs in the bone marrow and describes how this difference affects the clinical values of CTCs and DTCs, such as metastasis and recurrence. We suggest that DTCs remaining in the bone marrow after therapy can be used as a superior marker in comparison with CTCs to define patients with an unfavourable prognosis and may therefore be a potential prognostic factor and therapeutic target for cancer therapy.


| CANCER CELL DISSEMINATION IS AN EARLY EVENT
Cancer cell dissemination has long been considered to be a late event in tumour development. However, accumulating evidence indicates that cancer cells spread much earlier than was previously believed, 8 even before the primary tumour is detected. 9 Tumour cells are frequently detected in the blood and bone marrow of cancer patients who have no clinical or even histopathologic signs of metastasis. 10 The variability in detection rates is likely due to differences in selection criteria and methodologies (Table 1). Recent technological advances have greatly improved CTC detection methods. An advanced unique microfluidic platform (CTC-Chip) was found to identify CTCs in the peripheral blood of more than 90% of patients with metastatic lung, prostate, pancreatic, breast cancer and colon cancer and did not detect CTCs in the healthy control.
In addition, CTCs were isolated in 100% of patients with earlystage prostate cancer using the same platform, 11,12 indicating that the dissemination of cancer cells into the circulation may occur randomly. CTCs that home to the bone marrow are also detected in patients with pre-invasive lesions, suggesting that bloodborne dissemination is also an early event. 12 Given the much lower incidences of metastasis, the correlation between CTCs, DTCs and metastasis remains elusive. To date, the detection of CTCs and DTCs remains a challenging diagnostic approach and prognostic biomarker, not only as a result of methodological limitations but also because the heterogeneity among CTCs and DTCs in bone marrow compromises their ability to predict the metastatic behaviours.
Neither CTC status nor DTC status has been included in routine clinical analysis. 13

| BONE MARROW IS A RESERVOIR OF DISSEMINATED TUMOUR CELLS
Bone marrow is a critical site of immune cell development and erythropoiesis. The bone marrow parenchyma includes hematopoietic stem cells and hematopoietic progenitor cells. The stroma, which is composed of stromal stem cells, extracellular matrix and several types of secreted cytokines, is highly vascular and enriched with numerous blood vessels and capillaries. 14 Bone marrow is also a niche for mature plasma cells and memory T cells. 15 Bone marrow displays structural and functional features resembling a secondary lymphoid organ, providing appropriate support for T cells. Accumulated evidence demonstrates that, in addition to the hematopoietic progenitor cells, bone marrow contains various immune cells, including regulatory T cells, conventional T cells, B cells, dendritic cells, natural killer T (NKT) cells, neutrophils, myeloid-derived suppressor cells and mesenchymal stem cells. 14 Bone marrow diseases, including leukaemia, lymphoma, multiple myeloma anaemia and other life-threatening diseases, lead to an abnormality in the production of mature blood cells. Bone marrow is a preferred metastatic site for several solid tumours, such as breast cancer, lung cancer, prostate cancer and others. 16 Bone marrow also represents a sanctuary site for DTCs derived from various additional types of epithelial tumours. 16 The presence of DTCs in the bone marrow is associated with not only bone metastasis but also the development of distant tumours. DTCs in the bone marrow may re-enter the vasculature and disseminate secondarily throughout the body. 17,18 Although the dissemination of cancer cells into the circulation and the bone marrow is an efficient process, cancer metastasis is an

| BON E MARROW FOR MS A PREMETASTATIC NICHE FOR DISSEMINATED TUMOUR CELLS
Secondary cancer growths do not occur randomly. 23 Stephen Paget's "seed and soil" hypothesis proposed that the homing and settlement of cancer cells depend on the fertile "soil" provided by a given microenvironment. Bone marrow is a major target organ for metastasis, providing a fertile "soil" for circulating tumour cells to settle and repopulate. 24 The fertile "soil" is sometimes formed before the arrival of circulating tumour cells, in which case it is termed a "premetastatic niche." Cells in this favourable microenvironment or "premetastatic niche" secrete many cytokines and chemokines, attracting primary tumour cells to the niche as well as supporting their subsequent colonization. The formation of the premetastatic niche is influenced by the primary cancer. 25 Recent studies suggest that primary tumour cell-derived extracellular vesicles (EV) contribute to distant metastasis effectively. EV carried miR-122 suppress glucose uptake of microenvironmental cells by downregulating the glycolytic enzyme pyruvate kinase. 26 Tumour exosomal small nuclear RNAs remodel the lung metastatic niche by activating alveolar epithelial TLR3 to recruit neutrophil. 27 The premetastatic niche is defined as a microenvironment that is highly proinflammatory, adhesive, vascular, proproliferative and chemotactic. Integrins on both tumour cells and the supporting stromal cells in the bone marrow, such as osteoclasts, inflammatory cells and bone marrow stromal cells, play key roles in enhancing the localization of cancer cells to the bone marrow. 28 The interaction of integrins and their ligands allows for the mobilization and adhesion of cancer cells to the bone marrow stromal cells (αvβ3, α5β1, α4β1), [29][30][31] osteoclasts (αvβ3), 32,33 platelets (αvβ3, α1β3), 34 bone marrow hematopoietic progenitor cells (α4β1) 28,35 and endothelial cells (α1β1, α2β1). 36 Chemokines such as SDF-1, 37,38 S100A8 and S100A9, 39 which are produced by bone marrow-derived cells (BMDCs) or primary cancer cells, elicit the accumulation of cancer cells, endothelial cells and macrophages in the bone marrow. 39 Cancer-induced stress elicits BMDCs, such as bone marrowderived hematopoietic progenitor cells, and the mobilization of stromal stem cells from the bone marrow into the primary cancer microenvironment. 40 Bone marrow-derived mesenchymal stem cells have the potential to develop into cancer-associated fibroblasts (CAFs). 41 Il-6 and GM-CSF were identified as the key factors released from CAFs that promote tumour-associated macrophages infiltration in the premetastatic niche. 42

| Bone marrow is a dormant niche for DTCs
Metastasis is an inefficient process. Although the dissemination of cancer cells occurs at an early stage of cancer, many cancer cells delivered into the circulation either die or go into a dormant state. 52 The following three types of gene expression signatures have been identified in CTCs: low-proliferative signatures, biphenotypic signatures, in which both epithelial and mesenchymal markers are expressed, and epithelial-stromal interface signatures, in which igfbp5 is expressed. 53 Due to the short half-life of CTCs in circulation, which persist for only 1-2 hours, and due to apoptotic CTCs, the detection and prognostic value of CTCs in circulation has been unclear. Most CTCs die in circulation as a result of shear stress and/or anoikis. 54,55 Contact with integrins and exposure to bone-derived cytokines in the bone marrow can reduce proapoptotic signalling. 56,57 The persistence of DTCs in the bone marrow during follow-up is an indicator of the prognosis. 58 Up to 40% of patients diagnosed with nonmetastatic breast cancer still have a significant risk of relapse, even after the complete surgical removal of the tumour, most likely because of the existence of DTCs. 51,59 DTCs in the bone marrow are usually considered to be nonproliferative and are believed to be the source of metastasis, independent of the primary tumour site and the pattern of overt metastases. 10 More than 80% of patients with solid tumours harbour Ki67-negative DTCs in the bone marrow. 60 DTCs in the bone marrow exhibit very low or even no detectable pAKT levels. 61  osteoclasts. 65 TGF-β derived from osteoclasts was also shown to play roles in maintaining the quiescent state of cancer cells in the bone marrow. 65 The HSC niche may act as a permissive site for DTCs to escape from source of stress. DTCs may reside in close proximity to osteoblasts while expressing high levels of Axl, one of the tyrosine kinase receptors for growth arrest-specific 6 (GAS6

| Immuno-mediated dormancy in bone marrow
The process of tumour immuno-editing includes the following three key phases: elimination, equilibrium and escape. 68 Figure 2). [90][91][92][93][94] The synergistic effect of Snail and β-catenin confers cancer cells the ability to survive during dissemination and invasion. 95 Additionally, DTCs often display attributes of a stem cell-like phenotype (CD44 high /CD24 low ) related to self-renewal and angiogenesis. [96][97][98][99] This more plastic phenotype may be endowed by the primary tumour and clonally selected for during the process of homing, or promoted by the bone marrow niche as a stem cell niche to induce terminally differentiated cells to become stemlike. 46 The mesenchymal phenotype may be a transient state of DTCs in the bone marrow, keeping them dormant and resistant to radiotherapy/chemotherapy. 100 The overexpression of unfolded protein response (UPR) proteins and protein disulphide-isomerase was observed in DTCs from bone marrow, suggesting that DTCs may resist the stress attributed to UPR phenotype. 96  status. 105 This discordance may be important for determining which patients will benefit from endocrine and/or HER2 targeted therapy.

| DTCs in the bone marrow displayed an osteoblast-like or osteoclast-like phenotype
Patients lacking ER or HER2 expression on the primary cancer but showing ER-positive or HER2-positive DTCs may benefit from an endocrine and/or HER2-targeted therapy. This discordance may explain the failure rates seen in conventional endocrine adjuvant therapy for patients with DTCs that were ERα negative despite the presence of ERα positive primary tumours. 106 Approximately 90% of analysed breast cancer patients with HER3 activation were found to exhibit very low or even no detectable levels of pAKT S473, suggesting that these cells might have fallen into dormancy. 61

| DTC IN BONE MARROW MAY BE A PR OGNOSTIC BIOMARKE R FOR RELAPSE AND A THERAPEUTIC TARGET FOR CANCER
Lymphatic metastasis has long been considered to be the primary route of metastasis, partly due to the accessibility of the lymph nodes. However, haematogenous metastasis is becoming increasingly recognized and confirmed because of the detection of CTCs. Lymphnode involvement does not accurately predict the haematogenous dissemination of cancer cells, nor is haematogenous dissemination necessarily associated with lymph-node involvement. 59  CellSearch ® system is already applied in tracking the CTCs in cancer patients. 110 Clinically, liquid bioscopy helps to find DTCs in the bone marrow of patients with various types of solid tumours. 16 CTCs and DTCs have been reported as independent prognostic markers that impact the progression-free survival (PFS) and overall survival (OS). 13 The number of CTCs is also correlated with serum levels of other markers, such as the prostate cancer biomarker, PSA. 49 However, the inconsistency of these markers and the short half-life of CTCs limit their application as diagnostic and prognostic biomarkers in the clinical setting. It has been considered that CTCs are singly suspended in the circulation, but the amount of CTC clusters can be underestimated due to the lack of appropriate detection methods. 111 The survival rates of CTCs are highly CTCs were detected in 10% of the patients, and DTCs were detected in 14% of the same patients. 115 As DTCs in the bone marrow display a more malignant transformed phenotype, and the colonization of CTCs in the bone marrow is more like a selective process, DTCs in bone marrow are expected to be a superior marker for predicting overall survival, compared to CTCs. However,this conclusion is controversial. [114][115][116][117] Some studies have demonstrated a superior performance of DTCs in predicting overall survival in patients with cancer. 118 In contrast, other studies reported that DTCs were associated with bone metastasis (P = 0.0001) but not with a poorer overall survival. 116 At this time, DTCs have not been recommended as a substitute for CTCs as the prognostic biomarker, because bone marrow aspiration is invasive and uncomfortable for patients. However, the presence of bone marrow DTCs in patients with primary breast cancer is associated with a shorter relapse-free survival and locoregional relapse, although it is not an independent prognostic factor. 58,119 The persistence of DTCs during follow-up has been shown to significantly predict the increased risk for subsequent relapse and death. 120 The importance of histologic or genetic subclassification of cancer lies principally in the tumour biology, especially the response to therapy and the outcome. DTC status has been included in the new American Joint Committee on cancer classification. 13 The presence of DTCs identified (at median 85 months follow-up) a subgroup of luminal A patients characterized by high expression levels of ER-related genes and low expression of the HER2 cluster and proliferation-associated genes. This subgroup was found to have particularly poor outcomes (P = 0.008); however, this finding was not apparent for other tumour subtypes. 121 DTCs, but not CTCs, are associated with DFS in node-negative patients. 115 Currently, no diagnostic tools are available to monitor the response after the completion of adjuvant treatment to identify patients who need secondary adjuvant therapy due to persistent tumour cell load.
The persistence of DTCs in the bone marrow after a disease-free follow-up interval indicates that DTCs in the bone marrow may be used as a surrogate marker to predict the risk of relapse, especially to identify patients with a poor response to therapy. The reevaluation of bone marrow status may be a promising tool because the presence of DTCs is a possible surrogate marker for persistent MRD.
Studies have shown that the persistence of DTCs in the bone marrow of patients with primary breast cancer after conventional adjuvant therapy is associated with a poor prognosis. 106 The existence of dormant DTCs in the bone marrow increases the risk of late relapse. When patients were followed for years, DTCs in the bone marrow were found to be a superior marker for predicting relapse.
The persistence of DTCs in the bone marrow during follow-up significantly predicted the increased risk for subsequent relapse and could be used as an indicator for secondary treatment intervention. 120 Combining the data from various laboratories, it is estimated that before treatment, the positivity of DTCs in the bone marrow is approximately 30%. 122 Treatment with clodronate has made a significant improvement in OS and significantly reduced the incidence of bone metastasis. 128 Preventing dormant DTCs in the bone marrow from reactivating cell cycling or kicking dormant cancer cells back into circulation may be strategies for preventing the relapse of metastatic disease.
β-adrenergic receptor antagonists, which interfere with proliferative re-activation norepinephrine (NE) signalling, may reduce cancer relapse or slow disease progression. 129 One potential strategy is finding a way to inhibit E-selectin, which could limit the ability of the cancer cells to travel into the bone and resurge as metastatic cancer. A combination of the E-selectin inhibitor GMI-1271, daunorubicin and cytarabine, was shown to result in a greater depletion of AML from the bone marrow. 130 Moreover, GMI-1271 was reported to enhance the response to chemotherapy. Bone marrow transplantation could also be a good choice after the surgery or chemotherapy.

| CONCLUSION
Dissemination of cancer cells is considered to be an early and random event in the process of cancer progression as detection methods have been greatly improved. DTCs in the bone marrow may be endowed with particular characteristics that are different from CTCs in the circulation by the special environment. These dormant, mesenchymal, osteoblast-like or osteoclast-like signatures may provide superior markers to CTCs for predicting metastasis or relapse of cancer and provide potential therapeutic targets for therapy.

CONF LICTS OF INTEREST
The authors declare that they have no conflict of interests.