Cells in the Polyaneuploid Cancer Cell State are Pro-Metastatic.

There remains a large need for a greater understanding of the metastatic process within the prostate cancer field. Our research aims to understand the adaptive - ergo potentially metastatic - responses of cancer to changing microenvironments. Emerging evidence has implicated a role of the Polyaneuploid Cancer Cell (PACC) state in metastasis, positing the PACC state as capable of conferring metastatic competency. Mounting in vitro evidence supports increased metastatic potential of cells in the PACC state. Additionally, our recent retrospective study of prostate cancer patients revealed that PACC presence in the prostate at the time of radical prostatectomy was predictive of future metastatic progression. To test for a causative relationship between PACC state biology and metastasis, we leveraged a novel method designed for flow-cytometric detection of circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) in subcutaneous, caudal artery, and intracardiac mouse models of metastasis. This approach provides both quantitative and qualitative information about the number and PACC-status of recovered CTCs and DTCs. Collating data from all models, we found that 74% of recovered CTCs and DTCs were in the PACC state. In vivo colonization assays proved PACC populations can regain proliferative capacity at metastatic sites following dormancy. Additional direct and indirect mechanistic in vitro analyses revealed a PACC-specific partial Epithelial-to-Mesenchymal-Transition phenotype and a pro-metastatic secretory profile, together providing preliminary evidence that PACCs are mechanistically linked to metastasis.

Multiparameter flow cytometric detection and analysis of rare cells in in vivo models of cancer metastasis.

Rapid and reliable circulating tumor cell (CTC) and disseminated tumor cell (DTC) detection are critical for rigorous evaluation of in vivo metastasis models. Clinical data show that each step of the metastatic cascade presents increasing barriers to success, limiting the number of successful metastatic cells to fewer than 1 in 1,500,000,000. As such, it is critical for scientists to employ approaches that allow for the evaluation of metastatic competency at each step of the cascade. Here, we present a flow cytometry-based method that enables swift and simultaneous comparison of both CTCs and DTCs from single animals, enabling evaluation of multiple metastatic steps within a single model system. We present the necessary gating strategy and optimized sample preparation conditions necessary to capture CTCs and DTCs using this approach. We also provide proof-of-concept experiments emphasizing the appropriate limits of detection of these conditions. Most importantly, we successfully recover CTCs and DTCs from murine blood and bone marrow. In Supplemental materials, we expand the applicability of our method to lung tissue and exemplify a potential multi-plexing strategy to further characterize recovered CTCs and DTCs. This approach to multiparameter flow cytometric detection and analysis of rare cells in in vivo models of metastasis is reproducible, high throughput, broadly applicable, and highly adaptable to a wide range of scientific inquiries. Most notably, it simplifies the recovery and analysis of CTCs and DTCs from the same animal, allowing for a rapid first look at the comparative metastatic competency of various model systems throughout multiple steps of the metastatic cascade.

Circulating tumor cells in neuroblastoma: Current status and future perspectives.

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 10% to 20% of deaths of pediatric malignancies. Due to the poor prognosis and significant biological heterogeneity of neuroblastoma, it is essential to develop personalized therapeutics and monitor treatment response. Circulating tumor cells (CTCs), as one of the important analytes for liquid biopsy, could facilitate response assessment and outcome prediction for patients in a non-invasive way. Several methods and platforms have been used for the enrichment and detection of CTCs. The enumeration of CTCs counts and evaluation of tumor-specific mRNA transcript levels could provide prognostic information at diagnosis, during or after chemotherapy, and during the process of disease progression. So far, studies into neuroblastoma CTCs are only in the preliminary stages. The quality-controlled large prospective cohort studies are needed to evaluate the clinical significance and statistical rigor of CTC detection methods. Moreover, there remains a lot to be explored and investigated in genotyping characterization of neuroblastoma (NB) CTCs and construction of in-vitro or in-vivo functional models. CTCs and circulating tumor DNA (ctDNA) analysis will be complementary in understanding tumor heterogeneity and evolution over the course of therapy for patients with NB in the future.

Mini-Review: Can the Metastatic Cascade Be Inhibited by Targeting CD147/EMMPRIN to Prevent Tumor Recurrence?

Solid tumors metastasize very early in their development, and once the metastatic cell is lodged in a remote organ, it can proliferate to generate a metastatic lesion or remain dormant for long periods. Dormant cells represent a real risk for future tumor recurrence, but because they are typically undetectable and insensitive to current modalities of treatment, it is difficult to treat them in time. We describe the metastatic cascade, which is the process that allows tumor cells to detach from the primary tumor, migrate in the tissue, intravasate and extravasate the lymphatics or a blood vessel, adhere to a remote tissue and eventually outgrow. We focus on the critical enabling role of the interactions between tumor cells and immune cells, especially macrophages, in driving the metastatic cascade, and on those stages that can potentially be targeted. In order to prevent the metastatic cascade and tumor recurrence, we would need to target a molecule that is involved in all of the steps of the process, and evidence is brought to suggest that CD147/EMMPRIN is such a protein and that targeting it blocks metastasis and prevents tumor recurrence.

Gene expression predicts dormant metastatic breast cancer cell phenotype.

BACKGROUND: Breast cancer can recur months to decades after an initial diagnosis and treatment. The mechanisms that control tumor cell dormancy remain poorly understood, making it difficult to predict which patients will recur and thus benefit from more rigorous screening and treatments. Unfortunately, the extreme rarity of dormant DTCs has been a major obstacle to their study. METHODS: To overcome this challenge, we developed an efficient system to isolate and study rare dormant breast cancer cells from metastatic organs including bones, which represent a major site of metastasis. After isolation of cells from the long bones, we used single cell RNA-sequencing (scRNA-seq) to profile proliferative and dormant PyMT-Bo1 breast cancer cells. We also compared this signature to dormant versus proliferative tumor cells isolated from the lungs. Finally, we compared our dormant signature to human datasets. RESULTS: We identified a group of genes including Cfh, Gas6, Mme and Ogn that were highly expressed in dormant breast cancer cells present in the bone and lung. Expression of these genes had no impact on dormancy in murine models, but their expression correlated with disease-free survival in primary human breast cancer tumors, suggesting that these genes have predictive value in determining which patients are likely to recur. CONCLUSIONS: Dormant breast cancer cells exhibit a distinct gene expression signature regardless of metastatic site. Genes enriched in dormant breast cancer cells correlate with recurrence-free survival in breast cancer patients.

The PI3K/mTOR inhibitor Gedatolisib eliminates dormant breast cancer cells in organotypic culture, but fails to prevent metastasis in preclinical settings.

Dormant, disseminated tumor cells (DTCs) are thought to be the source of breast cancer metastases several years or even decades after initial treatment. To date, a selective therapy that leads to their elimination has not been discovered. While dormant DTCs resist chemotherapy, evidence suggests that this resistance is driven not by their lack of proliferation, but by their engagement of the surrounding microenvironment, via integrin-ß1-mediated interactions. Because integrin-ß1-targeted agents have not been translated readily to the clinic, signaling nodes downstream of integrin-ß1 could serve as attractive therapeutic targets in order to sensitize dormant DTCs to therapy. By probing a number of kinases downstream of integrin-ß1, we determined that PI3K inhibition with either a tool compounds or a compound (PF-05212384; aka Gedatolisib) in clinical trials robustly sensitizes quiescent breast tumor cells seeded in organotypic bone marrow cultures to chemotherapy. These results motivated the preclinical study of whether Gedatolisib-with or without genotoxic therapy-would reduce DTC burden and prevent metastases. Despite promising results in organotypic culture, Gedatolisib failed to reduce DTC burden or delay, reduce or prevent metastasis in murine models of either triple-negative or estrogen receptor-positive breast cancer dissemination and metastasis. This result held true whether analyzing Gedatolisib on its own (vs. vehicle-treated animals) or in combination with dose-dense doxorubicin and cyclophosphamide (vs. animals treated only with dose-dense chemotherapies). These data suggest that PI3K is not the node downstream of integrin-ß1 that confers chemotherapeutic resistance to DTCs. More broadly, they cast doubt on the strategy to target PI3K in order to eliminate DTCs and prevent breast cancer metastasis.

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

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

Minimal residual disease in high-risk neuroblastoma shows a dynamic and disease burden-dependent correlation between bone marrow and peripheral blood.

Neuroblastoma (NB) is the most common extracranial solid tumor in children and originates from sympathoadrenal or Schwann cell precursors derived from neural crest. These neural crest derivatives also constitute the hematopoietic and mesenchymal stem cells in bone marrow (BM) that is the most frequent site of NB metastasis and relapse. In NB patients, NB cells have been pathologically detected in BM and peripheral blood (PB), and minimal residual disease (MRD) in BM and PB (BM-MRD and PB-MRD) can be monitored by quantitating several sets of NB-associated mRNAs (NB-mRNAs). Although previous studies have shown varying degrees of correlation between BM-MRD and PB-MRD, the underlying factors and/or mechanisms remains unknown. In the present study, we determined the levels of BM-MRD and PB-MRD by quantitating seven NB-mRNAs in 133 pairs of concurrently collected BM and PB samples from 19 high-risk NB patients with clinical disease evaluation, and examined their correlation in overall and subgroups of sample pairs. The levels of BM-MRD and PB-MRD were moderately (r = 0.418, p < 0.001) correlated with each other in overall sample pairs. The correlation became strong (r = 0.725, p < 0.001), weak (r = 0.284, p = 0.008), and insignificant (p = 0.194) in progression, stable, and remission subgroups of sample pairs, respectively. It also became stronger in subgroups of sample pairs with poor treatment responses and poor prognostic factors. Present study suggests that MRD in high-risk NB shows a dynamic and disease burden-dependent correlation between BM and PB.

Molecular pathology underlying the robustness of cancer stem cells.

Intratumoral heterogeneity is tightly associated with the failure of anticancer treatment modalities including conventional chemotherapy, radiation therapy, and molecularly targeted therapy. Such heterogeneity is generated in an evolutionary manner not only as a result of genetic alterations but also by the presence of cancer stem cells (CSCs). CSCs are proposed to exist at the top of a tumor cell hierarchy and are undifferentiated tumor cells that manifest enhanced tumorigenic and metastatic potential, self-renewal capacity, and therapeutic resistance. Properties that contribute to the robustness of CSCs include the abilities to withstand redox stress, to rapidly repair damaged DNA, to adapt to a hyperinflammatory or hyponutritious tumor microenvironment, and to expel anticancer drugs by the action of ATP-binding cassette transporters as well as plasticity with regard to the transition between dormant CSC and transit-amplifying progenitor cell phenotypes. In addition, CSCs manifest the phenomenon of metabolic reprogramming, which is essential for maintenance of their self-renewal potential and their ability to adapt to changes in the tumor microenvironment. Elucidation of the molecular underpinnings of these biological features of CSCs is key to the development of novel anticancer therapies. In this review, we highlight the pathological relevance of CSCs in terms of their hallmarks and identification, the properties of their niche-both in primary tumors and at potential sites of metastasis-and their resistance to oxidative stress dependent on system xc (-).

Interleukins as Mediators of the Tumor Cell-Bone Cell Crosstalk during the Initiation of Breast Cancer Bone Metastasis.

Patients with advanced breast cancer are at high risk of developing bone metastasis. Despite treatment advances for primary breast cancer, metastatic bone disease remains incurable with a low relative survival. Hence, new therapeutic approaches are required to improve survival and treatment outcome for these patients. Bone is among the most frequent sites of metastasis in breast cancer. Once in the bone, disseminated tumor cells can acquire a dormant state and remain quiescent until they resume growth, resulting in overt metastasis. At this stage the disease is characterized by excessive, osteoclast-mediated osteolysis. Cells of the bone microenvironment including osteoclasts, osteoblasts and endothelial cells contribute to the initiation and progression of breast cancer bone metastasis. Direct cell-to-cell contact as well as soluble factors regulate the crosstalk between disseminated breast cancer cells and bone cells. In this complex signaling network interleukins (ILs) have been identified as key regulators since both, cancer cells and bone cells secrete ILs and express corresponding receptors. ILs regulate differentiation and function of bone cells, with several ILs being reported to act pro-osteoclastogenic. Consistently, the expression level of ILs (e.g., in serum) has been associated with poor prognosis in breast cancer. In this review we discuss the role of the most extensively investigated ILs during the establishment of breast cancer bone metastasis and highlight their potential as therapeutic targets in preventing metastatic outgrowth in bone.

NK cells-directed therapies target circulating tumor cells and metastasis.

Metastasis is the major cause of cancer-related deaths. Invasive primary cancers often metastasize after circulating tumor cells (CTCs) enter the bloodstream or lymph node to colonize adjacent tissue or distant anatomical locations. CTCs interact with immune cells and metastatic microenvironments, survival signaling, and chemotherapeutic resistance. Among immune cells, natural killer (NK) cells can, directly and indirectly, interact with CTCs to control cancer metastasis. Understanding the molecular mechanisms that drive NK cells mediated recognition and elimination of CTCs may pave the way for a new generation of anti-CTC molecularly targeted immunotherapies. In this review, we will discuss i) the role of CTCs in metastases, ii) CTCs in the context of the tumor microenvironment, iii) CTCs immune escape, and finally, iv) the potentials of NK cell-based therapies alone, or in combination with nanomedicine for targeted-immunotherapies of metastatic diseases.

When a House Is Not a Home: A Survey of Antimetastatic Niches and Potential Mechanisms of Disseminated Tumor Cell Suppression.

Over the last four decades, the cancer biology field has concentrated on cellular and microenvironmental drivers of metastasis. Despite this focus, mortality rates upon diagnosis of metastatic disease remain essentially unchanged. Would a small change in perspective help? Knowing what constitutes an inhospitable, rather than hospitable, microenvironment could provide the inspiration necessary to develop better therapies and preventative strategies. In this review, we canvas the literature for hints about what characteristics four common antimetastatic niches-skeletal muscle, spleen, thyroid, and yellow bone marrow-have in common. We posit that thorough molecular and mechanistic characterization of antimetastatic tissues may inspire reimagined therapies that inhibit metastatic development and/or progression in an enduring manner.

Metastatic Colonization: Escaping Immune Surveillance.

Cancer immunotherapy has shifted the paradigm in cancer therapy by revitalizing immune responses against tumor cells. Specifically, in primary tumors cancer cells evolve in an immunosuppressive microenvironment, which protects them from immune attack. However, during tumor progression, some cancer cells leave the protective tumor mass, disseminating and seeding secondary organs. These initial disseminated tumor cells (DTCs) should potentially be susceptible to recognition by the immune system in the new host tissues. Although Natural Killer or T cells eliminate some of these DTCs, a fraction escape anti-tumor immunity and survive, thus giving rise to metastatic colonization. How DTCs interact with immune cells and the underpinnings that regulate imperfect immune responses during tumor dissemination remain poorly understood. Uncovering such mechanisms of immune evasion may contribute to the development of immunotherapy specifically targeting DTCs. Here we review current knowledge about systemic and site-specific immune-cancer crosstalk in the early steps of metastasis formation. Moreover, we highlight how conventional cancer therapies can shape the pre-metastatic niche enabling immune escape of newly arrived DTCs.

Circulating tumor cells as Trojan Horse for understanding, preventing, and treating cancer: a critical appraisal.

Circulating tumor cells (CTCs) are regarded as harbingers of metastases. Their ability to predict response to therapy, relapse, and resistance to treatment has proposed their value as putative diagnostic and prognostic indicators. CTCs represent one of the zeniths of cancer evolution in terms of cell survival; however, the triggers of CTC generation, the identification of potentially metastatic CTCs, and the mechanisms contributing to their heterogeneity and aggressiveness represent issues not yet fully deciphered. Thus, prior to enabling liquid biopsy applications to reach clinical prime time, understanding how the above mechanistic information can be applied to improve treatment decisions is a key challenge. Here, we provide our perspective on how CTCs can provide mechanistic insights into tumor pathogenesis, as well as on CTC clinical value. In doing so, we aim to (a) describe how CTCs disseminate from the primary tumor, and their link to epithelial-mesenchymal transition (EMT); (b) trace the route of CTCs through the circulation, focusing on tumor self-seeding and the possibility of tertiary metastasis; (c) describe possible mechanisms underlying the enhanced metastatic potential of CTCs; (d) discuss how CTC could provide further information on the tissue of origin, especially in cancer of unknown primary origin. We also provide a comprehensive review of meta-analyses assessing the prognostic significance of CTCs, to highlight the emerging role of CTCs in clinical oncology. We also explore how cell-free circulating tumor DNA (ctDNA) analysis, using a combination of genomic and phylogenetic analysis, can offer insights into CTC biology, including our understanding of CTC heterogeneity and tumor evolution. Last, we discuss emerging technologies, such as high-throughput quantitative imaging, radiogenomics, machine learning approaches, and the emerging breath biopsy. These technologies could compliment CTC and ctDNA analyses, and they collectively represent major future steps in cancer detection, monitoring, and management.

Liquid Biopsy Based Single-Cell Transcriptome Profiling Characterizes Heterogeneity of Disseminated Tumor Cells from Lung Adenocarcinoma.

The advent of rapid and inexpensive sequencing technology allows scientists to decipher intra-tumor heterogeneity spatially and temporally for resolving the evolutionary history of tumor and the underlying mechanism. However, studies on characterizing heterogeneity of disseminated tumor cells (DTCs) in liquid biopsies are rare because of the rarity and low viability of DTCs as well as a large number of non-tumor cells. Here, high-throughput single-cell transcriptome sequencing technology and rare DTC enrichment method are employed to decipher the heterogeneity and distinct molecular signatures of DTCs in malignant pleural effusion (MPE) from lung adenocarcinoma. Single-cell transcriptomes of 8213 MPE-derived cells are acquired for bioinformatics analysis. In these cells from MPE, five main cell populations including tumor, mesothelial, monocyte, T and B cells are identified with specific markers for each group. Tumor cells present in MPE are further divided into four distinct subgroups that are found to be associated with immune response, cell proliferation, apoptosis, and cell adhesion, respectively. Based on the single-cell dataset of MPE-derived DTCs, 19 tumor-specific markers are identified that are also highly expressed at RNA and protein levels in tumor tissues as candidate markers for detection of extraordinarily rare circulating tumor cells in the blood.

Circulating and Disseminated Tumor Cells in Breast Carcinoma: Report from the Consensus Conference on Tumor Cell Dissemination during the 39th Annual Meeting of the German Society of Senology, Berlin, 27 June 2019.

Hematogenous dissemination of single tumor cells from the primary tumor is a common phenomenon in most solid malignancies. In breast cancer, presence of circulating tumor cells (CTCs) in the peripheral blood and disseminated tumor cells (DTCs) in bone marrow predicts poor clinical outcome, both in early and metastatic setting. Beyond that, persistence of CTCs/DTCs is associated with shorter relapse-free interval as well. Numerous studies have shown that these cells differ from tumor cells in the primary tumor with regard to hormone and HER2 receptor status and it has been hypothesized that some of them might be in fact cancer stem cells. Recently, the first positive study on CTC-based therapy interventions has been presented at the San Antonio Breast Cancer Symposium 2018, demonstrating that detection of CTCs may guide treatment decisions in metastatic HR-positive HER2-negative disease. In this review, we present the current state of evidence of tumor cell dissemination and discuss the implications for future trials.

Detection of disseminated tumor cells in bone marrow predict late recurrences in operable breast cancer patients.

BACKGROUND: Operable breast cancer patients may experience late recurrences because of reactivation of dormant tumor cells within the bone marrow (BM). Identification of patients who would benefit from extended therapy is therefore needed. METHODS: BM samples obtained pre- and post-surgery were previously analysed for presence of disseminated tumor cells (DTC) by a multimarker mRNA quantitative reverse-transcription PCR assay. Updated survival analyses were performed on all patient data (n = 191) and in a subgroup of patients alive and recurrence-free after 5 years (n = 156). DTC data were compared to the mitotic activity index (MAI) of the primary tumors. Median follow-up time was 15.3 years. RESULTS: Among the 191 patients, 49 (25.65%) experienced systemic relapse, 24 (49%) within 5-18 years after surgery. MAI and pre- and post-operative DTC status had significant prognostic value based on Kaplan-Meier analyses and multiple Cox regression in the overall patient cohort. With exclusion of patients who relapsed or died within 5 years from surgery, only pre-operative DTC detection was an independent prognostic marker of late recurrences. High MAI (≥10) did not predict late recurrences or disease-specific mortality. CONCLUSION: Pre-operative DTC detection, but not MAI status, predicts late recurrences in operable breast cancer.

Parallels between hematopoietic stem cell and prostate cancer disseminated tumor cell regulation.

The bone marrow is the primary site of hematopoiesis and the home for hematopoietic stem cells (HSCs) in adult mammals. Prostate cancer commonly metastasizes to the bone and forms bone metastases in almost all patients who die of the disease. Prostate cancer bone metastases are thought to develop after rare bone marrow disseminated tumor cells (DTCs) escape a dormant state and reactivate. Prostate cancer DTCs and normal HSCs have been shown to compete for residence in the bone marrow and share many of same regulatory mechanisms for survival, proliferation and homing. In this review, we highlight these parallels in order to help our readers use the literature in HSC and DTC biology to inform their research and generate hypotheses in both fields.

Minimal Residual Disease in Prostate Cancer.

Detection of minimal residual disease (MRD) in prostate cancer over several decades has greatly informed our understanding of dissemination and recurrence, but has not yet been routinely used in clinical care. Investigators have detected MRD by identification of prostate cancer cells in the bone marrow; termed disseminated tumor cells (DTCs) and blood; termed circulating tumor cells (CTCs). Various techniques including PSA-RT PCR, PSA immunocytochemistry, cytokeratin immunocytochemistry, and immune-magnetic depletion of hematopoietic cells followed by EPCAM based positive selection, have been used. Importantly, detection of DTCs correlates with recurrence. Research into prostate cancer CTCs has intensified recently, but their use in MRD evaluation has been more limited. Investigators are using semi-automated platforms to detect and begin to study prostate cancer CTCs in patients with no evidence of disease. PSA immunocytochemistry also detects CTCs and correlates with recurrence. Emerging technologies have the potential to greatly aid research in this exciting field.

The Different Routes to Metastasis via Hypoxia-Regulated Programs.

Hypoxia is linked to metastasis; however, how it affects metastatic progression is not clear due to limited consensus in the literature. We posit that this lack of consensus is due to hypoxia being studied using different approaches, such as in vitro, primary tumor, or metastasis assays in an isolated manner. Here, we review the pros and cons of in vitro hypoxia assays, highlight in vivo studies that inform on physiological hypoxia, and review the evidence that primary tumor hypoxia might influence the fate of disseminated tumor cells (DTCs) in secondary organs. Our analysis suggests that consensus can be reached by using in vivo methods of study, which also allow better modeling of how hypoxia affects DTC fate and metastasis.