Early Disseminated Research Articles

Breast Cancer Disseminated Tumor Cells: Do They Stay and Fight or Run and Hide?

Many solid tumors including breast cancer can exhibit early dissemination and dormancy-in which cancer cells spread early in the disease process and survive long periods without detectable growth. These early disseminated tumor cells sometimes reactivate and lead to incurable metastatic disease years or even decades after curative-intent therapy for the primary tumor. We are just beginning to understand the role of the immune system in this process in part because of improvements in immunocompetent models as well as technological advances such as single-cell genomics and spatial transcriptomics. In this issue of Cancer Research, Bushnell and colleagues showed that NK cells are important in this context. The authors found that disseminated tumor cells and quiescent cells express higher levels of MHC 1 but are resistant to NK-cell-mediated immunity. The proposed mechanism involves the STING pathway and transcription factors Sox2 and Bach1. As other studies have highlighted the importance of T-cell immunity, this work reaffirms the importance and diversity of immune regulation of dormancy and suggests the need for future studies to flesh out mechanistic details and predict when each type of immunity is most important. See related article by Bushnell et al., p. 3337.

Combined Single-Cell and Spatial Transcriptomics Reveal the Metabolic Evolvement of Breast Cancer during Early Dissemination.

Breast cancer is now the most frequently diagnosed malignancy, and metastasis remains the leading cause of death in breast cancer. However, little is known about the dynamic changes during the evolvement of dissemination. In this study, 65968cells from fourpatients with breast cancer and paired metastatic axillary lymph nodes are profiled using single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics. A disseminated cancer cell cluster with high levels of oxidative phosphorylation (OXPHOS), including the upregulation of cytochrome C oxidase subunit 6C and dehydrogenase/reductase 2, is identified. The transition between glycolysis and OXPHOS when dissemination initiates is noticed. Furthermore, this distinct cell cluster is distributed along the tumor's leading edge. The findings here are verified in three different cohorts of breast cancer patients and an external scRNA-seq dataset, which includes eightpatients with breast cancer and paired metastatic axillary lymph nodes. This work describes the dynamic metabolic evolvement of early disseminated breast cancer and reveals a switch between glycolysis and OXPHOS in breast cancer cells as the early event during lymph node metastasis.

ZFP281 drives a mesenchymal-like dormancy program in early disseminated breast cancer cells that prevents metastatic outgrowth in the lung.

Increasing evidence shows that cancer cells can disseminate from early evolved primary lesions much earlier than the classical metastasis models predicted. Here, we reveal at a single-cell resolution that mesenchymal-like (M-like) and pluripotency-like programs coordinate dissemination and a long-lived dormancy program of early disseminated cancer cells (DCCs). The transcription factor ZFP281 induces a permissive state for heterogeneous M-like transcriptional programs, which associate with a dormancy signature and phenotype in vivo. Downregulation of ZFP281 leads to a loss of an invasive, M-like dormancy phenotype and a switch to lung metastatic outgrowth. We also show that FGF2 and TWIST1 induce ZFP281 expression to induce the M-like state, which is linked to CDH1 downregulation and upregulation of CDH11. We found that ZFP281 not only controls the early dissemination of cancer cells but also locks early DCCs in a dormant state by preventing the acquisition of an epithelial-like proliferative program and consequent metastases outgrowth.

STAT3 phosphorylation at Ser727 and Tyr705 differentially regulates the EMT\u2013MET switch and cancer metastasis

Epithelial–mesenchymal transition (EMT)/mesenchymal–epithelial transition (MET) processes are proposed to be a driving force of cancer metastasis. By studying metastasis in bone marrow-derived mesenchymal stem cell (BM-MSC)-driven lung cancer models, microarray time-series data analysis by systems biology approaches revealed BM-MSC-induced signaling triggers early dissemination of CD133+/CD83+ cancer stem cells (CSCs) from primary sites shortly after STAT3 activation but promotes proliferation towards secondary sites. The switch from migration to proliferation was regulated by BM-MSC-secreted LIF and activated LIFR/p-ERK/pS727-STAT3 signaling to promote early disseminated cancer cells MET and premetastatic niche formation. Then, tumor-tropic BM-MSCs circulated to primary sites and triggered CD151+/CD38+ cells acquiring EMT-associated CSC properties through IL6R/pY705-STAT3 signaling to promote tumor initiation and were also attracted by and migrated towards the premetastatic niche. In summary, STAT3 phosphorylation at tyrosine 705 and serine 727 differentially regulates the EMT–MET switch within the distinct molecular subtypes of CSCs to complete the metastatic process.

Abstract 1150: Targeting PI3K inhibits ER-mammary tumor initiation and early dissemination

Abstract Despite recent advances in cancer treatment, including targeted therapies and immunotherapies, many women still die of breast cancer. To ultimately reduce breast cancer death, it is of urgent need to develop more effective prevention and early intervention strategies. Although tamoxifen and aromatase inhibitors decrease incidences of estrogen receptor positive (ER+) breast cancer, there is no effective agent for prevention of ER negative (ER-) breast cancer. In this study, we identified that PI3K pathway is significantly activated in premalignant ER- breast lesions compared to paired normal tissue from breast cancer patients. We therefore tested whether blocking PI3K pathway by genetic knockdown or using a clinically applicable PI3K inhibitor (GDC0941) can inhibit ER- tumor initiation and progression. Indeed, both genetic knockdown of PI3K and low dose GDC0941 intervention reversed the disorganized three-dimensional growth of semi-transformed human ER- mammary epithelial cells in vitro. More importantly, low dose GDC0941 treatment significantly delayed the tumor initiation in MMTV-neu mouse model which develops ER-HER2+ mammary tumors. Notably, low dose GDC0941 treatment did not show discernable adverse effects, including no significant changes in body weight and liver functions, no immuno-toxicity. Since PI3K activation is crucial for early disseminated tumor cells, we collected bone marrow from treated mice versus vehicle control group, and examined the impact of GDC0941 treatment on early dissemination of tumor cells into the bone marrow. Excitingly, we found that administration of low dose GDC0941 significantly decreased CD45-ErBb2+/CK8/18+ early disseminated mammary tumor cells into the bone marrow, indicating that low dose PI3K inhibitor not only delayed primary tumor initiation but also prevented tumor cell early dissemination in the bone marrow. Taken together, these data support the notion that using low dose targeting drug to block PI3K pathway is safe and promising for ER- breast cancer prevention. Citation Format: Yuan Zhang, Jingyang Wang, Yi Xiao, Victoria Seewaldt, Dihua Yu. Targeting PI3K inhibits ER-mammary tumor initiation and early dissemination [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 1150.

Abstract 6061: NR2F1 limits dissemination of early cancer mammary epithelial cells

Abstract Cancer cells have been found to disseminate during very early and sometimes asymptomatic stages of tumor progression. At secondary organs, early disseminated cancer cells (eDCCs) can enter a non-proliferative state for prolonged periods of time. Intriguingly, eDCCs also carry metastasis- initiating capacity. However, the mechanisms by which these early cancer cells complete all steps of metastasis including metastasis-initiating capacity are unknown. Here we report that the orphan nuclear receptor and lineage commitment regulator NR2F1 serves as an early barrier to early dissemination propelled by HER2 signaling. NR2F1 loss of function in HER2 early cancer cells induced cell motility, loss of E-cadherin junctions, disorganized laminin-V deposition, decreased β-catenin membrane localization and increased micro-invading cells with CK14+/CK18 phenotype. Importantly, upon NR2F1 depletion in HER2 early cancer acini, TWIST, ZEB1 and PRRX1 levels were upregulated. Our findings reveal a previously unrecognized function of NR2F1 as a suppressor of dissemination during early stages of breast tumor progression. Thus, therapies that restore and activate NR2F1 function might limit early dissemination and metastatic outgrowth. Citation Format: Maria Soledad Sosa, Nupura Kale, Nitisha Shrivastava, Carolina Rodriguez-Tirado, Maria Carlini, Jiayi Ji, Javier Bravo-Cordero. NR2F1 limits dissemination of early cancer mammary epithelial cells [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 6061.

Abstract SY23-01: Blood-borne tumor cell dissemination in cancer patients

Abstract SY23-01: Blood-borne tumor cell dissemination in cancer patients

Metastasis: Slipping Control

Metastasis: Slipping Control

Mechanism of early dissemination and metastasis in Her2+ mammary cancer.

Metastasis is the leading cause of cancer-related deaths; metastatic lesions develop from disseminated cancer cells (DCCs) that can remain dormant. Metastasis-initiating cells are thought to originate from a subpopulation present in progressed, invasive tumours. However, DCCs detected in patients before the manifestation of breast-cancer metastasis contain fewer genetic abnormalities than primary tumours or than DCCs from patients with metastases. These findings, and those in pancreatic cancer and melanoma models, indicate that dissemination might occur during the early stages of tumour evolution. However, the mechanisms that might allow early disseminated cancer cells (eDCCs) to complete all steps of metastasis are unknown. Here we show that, in early lesions in mice and before any apparent primary tumour masses are detected, there is a sub-population of Her2+p-p38lop-Atf2loTwist1hiE-cadlo early cancer cells that is invasive and can spread to target organs. Intra-vital imaging and organoid studies of early lesions showed that Her2+ eDCC precursors invaded locally, intravasated and lodged in target organs. Her2+ eDCCs activated a Wnt-dependent epithelial-mesenchymal transition (EMT)-like dissemination program but without complete loss of the epithelial phenotype, which was reversed by Her2 or Wnt inhibition. Notably, although the majority of eDCCs were Twist1hiE-cadlo and dormant, they eventually initiated metastasis. Our work identifies a mechanism for early dissemination in which Her2 aberrantly activates a program similar to mammary ductal branching that generates eDCCs that are capable of forming metastasis after a dormancy phase.

Early dissemination seeds metastasis in breast cancer.

Accumulating data suggest that metastatic dissemination often occurs early during tumour formation but the mechanisms of early metastatic spread have not yet been addressed. Here, we studied metastasis in a HER2-driven mouse breast cancer model and found that progesterone-induced signalling triggered migration of cancer cells from early lesions shortly after HER2 activation, but promoted proliferation in advanced primary tumour cells. The switch from migration to proliferation was regulated by elevated HER2 expression and increased tumour cell density involving miRNA-mediated progesterone receptor (PGR) down-regulation and was reversible. Cells from early, low-density lesions displayed more stemness features than cells from dense, advanced tumours, migrated more and founded more metastases. Strikingly, we found that at least 80% of metastases were derived from early disseminated cancer cells (DCC). Karyotypic and phenotypic analysis of human disseminated cancer cells and primary tumours corroborated the relevance of these findings for human metastatic dissemination.

Suppression of early hematogenous dissemination of human breast cancer cells to bone marrow by retinoic Acid-induced 2.

Regulatory pathways that drive early hematogenous dissemination of tumor cells are insufficiently defined. Here, we used the presence of disseminated tumor cells (DTC) in the bone marrow to define patients with early disseminated breast cancer and identified low retinoic acid-induced 2 (RAI2) expression to be significantly associated with DTC status. Low RAI2 expression was also shown to be an independent poor prognostic factor in 10 different cancer datasets. Depletion of RAI2 protein in luminal breast cancer cell lines resulted in dedifferentiation marked by downregulation of ERα, FOXA1, and GATA3, together with increased invasiveness and activation of AKT signaling. Functional analysis of the previously uncharacterized RAI2 protein revealed molecular interaction with CtBP transcriptional regulators and an overlapping function in controlling the expression of a number of key target genes involved in breast cancer. These results suggest that RAI2 is a new metastasis-associated protein that sustains differentiation of luminal breast epithelial cells. We identified downregulation of RAI2 as a novel metastasis-associated genetic alteration especially associated with early occurring bone metastasis in ERα-positive breast tumors. We specified the role of the RAI2 protein to function as a transcriptional regulator that controls the expression of several key regulators of breast epithelial integrity and cancer.

Cancer early dissemination: cancerous epithelial-mesenchymal transdifferentiation and transforming growth factor signalling

Contrary to the long believed hypothesis, it is now evident that breast cancer cells can disseminate from the early phases of the oncogenesis; and that such early disseminated cells sometimes survive at the sites of dissemination and may outgrow after a long latency of years and decades. For cancer cells to leave their origin, they must at least transiently loosen their adhesion with adjacent epithelial cells and stroma, and become motile while avoiding anoikis. Such processes resemble epithelial-mesenchymal transdifferentiation (EMT), which normally takes place in situations such as embryogenesis and wound healing. Interestingly, the occurrence of an EMT-like process in breast cancer cells has been implicated in the generation of cancer stem-like cells, in which TGFβ1 signalling often plays core roles. Here, I discuss the current knowledge regarding cancerous EMT and its signalling pathways with the aim to consider the possible mechanisms of early dissemination, and also the generation of cancer stem-like cells in mammary tumour.

Early tumor dissemination, but late metastasis: insights into tumor dormancy

The classical model of metastasis is that tumor cell dissemination occurs late in tumor development, after the primary tumor has grown, and that only then will tumor cells invade the local tissue, enter the blood or lymphatic vessels, and colonize new sites to cause metastases. However, evidence increasingly indicates that single tumor cells spread to distant sites much earlier than previously believed. In this issue of the JCI, Eyles and colleagues provide new insight into the mechanisms underlying early tumor cell dissemination, formation of metastases, and tumor immunosurveillance using transgenic mice that spontaneously develop melanomas of the uvea. The authors provide striking evidence that tumor cells start to disseminate during the initial steps of tumor development, that late appearing metastases arise from these early disseminated tumor cells, and that CD8+ T cells inhibit the growth of disseminated tumor cells, surprisingly, not by cytotoxic effects, but through cytostatic effects.

Abstract 2229: CD133+/CD44+ Cancer Stem Cells Represent a Disproportionately Large Fraction of Early Bone Disseminated Prostate Cancer Tumor Cells

Abstract Introduction: Nearly 30% of prostate cancer patients presenting with organ confined disease experience biochemical recurrence within 10 years of curative prostatectomy. A recent analysis of bone marrow aspirates reported the presence of disseminated tumor cells (DTCs) in 57% of patients with no evidence of disease following prostatectomy, suggesting that early bone homing prostate cancer DTCs may contribute to disease recurrence. Little is currently known about the biology of these cells. Procedures: Murine models were developed to isolate and study early bone homing prostate cancer disseminated tumor cells. Early dissemination was modeled through intracardiac injection of 1 × 106 prostate cancer cells of the PC3 cell line into SCID mice 15-24 hours prior to euthanasia. Spontaneous early tumor cell dissemination was modeled using subcutaneous prostate cancer tumors formed in SCID mice by injecting 5 × 105 DU145 cells in matrigel three weeks prior to euthanasia. Bone marrow was flushed from the femurs of euthanized mice and depleted of cells of hematopoietic-lineage using the Lineage Cell Depletion Kit (Miltenyi Biotec, 130-092-211). DTCs were identified in lineage depleted murine marrow using a FITC conjugated antibody for human leukocyte antigen (>90% of PC3 and DU145 cells are HLA-ABC+) and isolated by fluorescence-activated cell sorting (FACS). Cell surface expression of the cancer stem cell markers CD133 and CD44 on bone homing DTCs were analyzed by flow cytometry. Results: Prostate cancer DTCs were detected in murine bone marrow, representing 3.1% ± 0.9% (n=4-mice) and 1.2% ± 0.2% (n=4-mice) of lineage depleted femoral marrow cells from the intracardiac and subcutaneous models, respectively. Early bone disseminated PC3 cells in the intracardiac injection model demonstrated a large fraction of CD133+/CD44+ cancer stem cells (25.3% ± 4.2%, n=4). This is substantially greater than the 0.73% fraction found with the injected PC3 cells. Spontaneous early disseminated bone homing DTCs from subcutaneous DU145 tumors demonstrated similar results. A large fraction of DTCs were positive for both stem cell markers (17.3% ± 3.6%, n=4), considering that the injected DU145 cells only had a 0.28% fraction of CD133+/CD44+ cells. Conclusions: Early bone disseminated prostate cancer cells in murine xenograft models can be enriched by lineage depletion, identified by human leukocyte antigen, and subsequently isolated and characterized by flow cytometry. Early prostate cancer bone disseminations in both intracardiac injection and subcutaneous tumor models have a disproportionately large fraction of cancer stem cells identified using the surface markers CD133 and CD44. This suggests a proclivity for bone dissemination by CD133+/CD44+ prostate cancer stem cells and a possible role for these cells as DTCs responsible for biochemical failure after curative prostatectomy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2229.