Disseminated Breast Cancer Cells Research Articles

Sustained delivery of celecoxib from nanoparticles embedded in hydrogel injected into the biopsy cavity to prevent biopsy-induced breast cancer metastasis.

We have previously reported that protracted Cyclooxygenase-2 (COX-2) activity in bone marrow-derived cells (BMDCs) infiltrating into biopsy wounds adjacent to the biopsy cavity of breast tumors in mice promotes M2-shift of macrophages and pro-metastatic changes in cancer cells, effects which were suppressed by oral administration of COX-2 inhibitors. Thus, local control of COX-2 activity in the biopsy wound may mitigate biopsy-induced pro-metastatic changes. A combinatorial delivery system-thermosensitive biodegradable poly(lactic acid) hydrogel (PLA-gel) incorporating celecoxib-encapsulated poly(lactic-co-glycolic acid) nanoparticles (Cx-NP/PLA-gel)-was injected into the biopsy cavity of Py230 murine breast tumors to achieve local control of COX-2 activity in the wound stroma. A single intra-biopsy cavity injection of PLA-gel loaded with rhodamine-encapsulated nanoparticles (NPs) showed sustained local delivery of rhodamine preferentially to infiltrating BMDCs with minimal to no rhodamine uptake by the reticuloendothelial organs in mice. Moreover, significant reductions in M2-like macrophage density, cancer cell epithelial-to-mesenchymal transition, and blood vessel density were observed in response to a single intra-biopsy cavity injection of Cx-NP/PLA-gel compared to PLA-gel loaded with NPs containing no payload. Accordingly, intra-biopsy cavity injection of Cx-NP/PLA-gel led to significantly fewer metastatic cells in the lungs than control-treated mice. This study provides evidence for the feasibility of sustained, local delivery of payload preferential to BMDCs in the wound stroma adjacent to the biopsy cavity using a combinatorial delivery system to reduce localized inflammation and effectively mitigate breast cancer cell dissemination.

Abstract 5534: Needle biopsy induces pro-metastatic progression and systemic dissemination of estrogen receptor-positive breast cancer cells through the COX-2/PGE2 cascade

Abstract Breast cancer is the most commonly diagnosed malignancy in the U.S. and over two-thirds of invasive breast cancer cases are diagnosed early stage. However, 15-year survival rates for early-stage breast cancer remain in the range of 70-77%. Mounting evidence showed an increased risk of disease progression and mortality among early-stage breast cancer patients whose surgery was delayed over 60 days after the diagnostic biopsy. Yet, the mechanism(s) underlying the rapidly increased mortality due to delay of surgery after diagnosis remains unknown. Our cohort analyses of early-stage breast cancer patients reveal the emergence of a significantly rising mortality risk when the biopsy-to-surgery interval was extended beyond 53 days. Additionally, histology of post-biopsy tumors shows prolonged retention of a metastasis-permissive wound stroma dominated by M2-like macrophages capable of promoting cancer cell epithelial-to-mesenchymal transition and angiogenesis by secretion of transforming growth factor beta 1 (TGF-β1) and vascular endothelial growth factor (VEGF). We show that needle biopsy promotes systemic dissemination of cancer cells through a mechanism of sustained activation of the cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) cascade, which favors M2-polarization and its associated pro-metastatic changes, but are abrogated by oral treatment with COX-2 inhibitors in estrogen receptor-positive (ER+) syngeneic mouse tumor models. Therefore, we conclude that needle biopsy of ER+ breast cancer provokes progressive pro-metastatic changes, which may explain the mortality risk posed by surgery delay after diagnosis. Also, our data may provide a rationale for a pharmacologic strategy to inhibit the COX-2/PGE2 cascade in cases when delay of surgery is unavoidable. Citation Format: Hiroyasu Kameyama, Macall Leslie, Reese Simmons, Yunguang Sun, Misung Yi, Priya Dondapati, John F. Langenheim, Kar-Ming Fung, Inna Chervoneva, Hallgeir Rui, Takemi Tanaka. Needle biopsy induces pro-metastatic progression and systemic dissemination of estrogen receptor-positive breast cancer cells through the COX-2/PGE2 cascade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5534.

Abstract 5535: Intracellular negative regulators of RTK-Ras-ERK signaling alter breast cancer perception of metastatic niche-derived growth factors

Abstract Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype that commonly metastasizes to distant organs, such as the lung, resulting in the poor clinical outcomes. TNBC cells commonly overexpress epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK) which when bound by microenvironmental growth factors, results in the activation of downstream effector kinases ERK-AKT and initiates transcriptional programs that control cell fate. Tumor cells, however, simultaneously receive many growth factor inputs from their respective microenvironments, many of which stimulate RTKs other than EGFR, that converge at ERK and AKT resulting in different transcriptional and cell fate outputs. This raises questions as to how tumor cells perceive, decode, and respond to a complex set of microenvironmental signals and what the relevance of this process may be for disease pathogenesis and treatment. To this end, our study aimed to determine how disseminated breast cancer cells perceive and respond to a complex growth factor milieu upon seeding and engraftment in the lung. Using live-cell microscopy techniques and TNBC cells carrying biosensors for ERK and Akt signaling pathways, we monitored the dynamic signaling behaviors of single tumor cells, in real-time, as they adapted to the environment in living human lung tissue. Strikingly, we found that individual disseminated tumor cells are less responsive to growth factors once seeded in the lung, as compared to outside this tissue, despite the presence of RTK stimulating ligands. We showed that partial activation of EGFR is necessary for complete activation of ERK signaling by other growth factor-RTK pairs, and expression of downstream ERK target genes, whereas AKT signaling was unaffected. Using quantitative modeling and experimentation, we showed that reshaping of ERK signaling response dynamics occurred via dysregulation of negative regulators that are dominantly controlled by EGFR signaling. Together, these results provide novel insight into how tumor cells perceive and respond to complex microenvironmental signals, which has important implications for drug targeting strategies. Citation Format: Vaibhav Murthy, Cemal Erdem, Jeremy Copperman, Marc Birtwistle, Alexander Davies. Intracellular negative regulators of RTK-Ras-ERK signaling alter breast cancer perception of metastatic niche-derived growth factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5535.

Outlook and opportunities for engineered environments of breast cancer dormancy.

Dormant, disseminated breast cancer cells resist treatment and may relapse into malignant metastases after decades of quiescence. Identifying how and why these dormant breast cancer cells are triggered into outgrowth is a key unsolved step in treating latent, metastatic breast cancer. However, our understanding of breast cancer dormancy in vivo is limited by technical challenges and ethical concerns with triggering the activation of dormant breast cancer. In vitro models avoid many of these challenges by simulating breast cancer dormancy and activation in well-controlled, bench-top conditions, creating opportunities for fundamental insights into breast cancer biology that complement what can be achieved through animal and clinical studies. In this review, we address clinical and preclinical approaches to treating breast cancer dormancy, how precisely controlled artificial environments reveal key interactions that regulate breast cancer dormancy, and how future generations of biomaterials could answer further questions about breast cancer dormancy.

N-3 PUFAs synergistically enhance the efficacy of doxorubicin by inhibiting the proliferation and invasion of breast cancer cells.

Breast cancer stands as a prominent contributor to cancer-related fatalities among women globally, characterized by an unfavorable prognosis, low survival rates, and its conventional treatment approach involving chemotherapy. Doxorubicin (DOXO) represents a potent anti-tumor agent widely employed in combating breast cancer. Regrettably, a substantial proportion of patients eventually develop resistance to DOXO treatment, elevating the risk of relapse and adverse clinical outcomes. Omega-3 polyunsaturated fatty acids (n-3 PUFAs), recognized as essential components of the human diet, have exhibited considerable promise in targeting malignant cells, initiating apoptosis, and impeding tumor proliferation and metastatic dissemination. Combining these nutritional supplements, particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), with DOXO presents a compelling strategy to augment treatment efficacy. The present study was conducted employing a breast cancer cell line, MCF-7, to assess the synergistic potential of DHA, EPA, and DOXO. Remarkably, the combination treatment yielded a substantial increase in cytotoxicity compared to the administration of DOXO alone. Furthermore, an enhancement in the suppression of metastasis was evident in the combination treatment relative to the exclusive use of DOXO. Cell cycle analysis unveiled that cells subjected to the combination treatment exhibited a more pronounced arrest in the G1 phase, signifying the combination's heightened effectiveness in impeding cell progression into the doubling phase. Collectively, the amalgamation of n-3 polyunsaturated fatty acids (n-3 PUFAs) emerges as a potent strategy for enhancing the therapeutic potential of DOXO, effectively restraining the growth and dissemination of breast cancer cells.

Hydroxytyrosol Counteracts Triple Negative Breast Cancer Cell Dissemination via Its Copper Complexing Properties.

Polyphenols have gained increasing attention for their therapeutic potential, particularly in conditions like cancer, due to their established antioxidant and anti-inflammatory properties. Recent research highlights their ability to bind to transition metals, such as copper. This is particularly noteworthy given the key role of copper both in the initiation and progression of cancer. Copper can modulate the activity of kinases required for the epithelial-mesenchymal transition (EMT), a process fundamental to tumor cell dissemination. We have previously demonstrated the copper-binding capacity of oleuropein, a secoiridoid found in Olea europaea. In the present study, we investigated the effect of hydroxytyrosol, the primary oleuropein metabolite, on the metastatic potential of three triple-negative breast cancer cell lines (MDA-MB-231, MDA-MB-468, and SUM159). We found that hydroxytyrosol modulated the intracellular copper levels, influencing both the epithelial and mesenchymal markers, by downregulating copper-dependent AKT phosphorylation, a member of the EMT signaling cascade, through Western blot, RT-qPCR, and immunofluorescence. Indeed, by optical spectra, EPR, and in silico approaches, we found that hydroxytyrosol formed a complex with copper, acting as a chelating agent, thus regulating its homeostasis and affecting the copper-dependent signaling cascades. While our results bring to light the copper-chelating properties of hydroxytyrosol capable of countering tumor progression, they also provide further confirmation of the key role of copper in promoting the aggressiveness of triple-negative breast cancer cells.

Pleckstrin-2-promoted PPM1B degradation plays an important role in transforming growth factor-β-induced breast cancer cell invasion and metastasis.

Transforming growth factor-β (TGF-β) is known to promote breast cancer cell migration, invasion, and dissemination; however, the underlying molecular mechanisms are not yet well characterized. Here, we report that TGF-β induces pleckstrin-2 (PLEK2) expression by Smad3 and signal transducer and activator of transcription 3 (STAT3) activating PLEK2 promoter activity. Higher PLEK2 expression is associated with poor prognosis in breast cancer patients. Overexpression and knockout experiments in MDA-MB-231 and MCF-7 breast cancer cells revealed that PLEK2 promotes cell migration, invasion, and dissemination in 2D and 3D cell culture. Moreover, PLEK2 promotes metastasis of breast cancer cells in vivo. Pleckstrin-2 localizes to the cell membrane and cell protrusions following TGF-β treatment. Furthermore, inhibition of PI3K phosphorylation abolishes TGF-β- and PLEK2-induced cell invasion. The carboxyl-terminal PH domain of PLEK2 is critical for TGF-β- and PLEK2-induced Akt activation and plays an important role in cell invasion. Pleckstrin-2 interacts with PPM1B and promotes its ubiquitin-dependent degradation. The PLEK2-PPM1B axis utilizes nuclear factor-κB signaling to promote cell migration and invasion. Our data implicate the TGF-β-STAT3/Smad3-PLEK2-PPM1B signaling cascade in TGF-β-induced breast cancer cell migration and invasion. These findings suggest that PLEK2/PPM1B could represent novel targets for the intervention of breast cancer metastasis.

LOXL1 and LOXL4 are novel target genes of the Zn2+-bound form of ZEB1 and play a crucial role in the acceleration of invasive events in triple-negative breast cancer cells.

EMT has been proposed to be a crucial early event in cancer metastasis. EMT is rigidly regulated by the action of several EMT-core transcription factors, particularly ZEB1. We previously revealed an unusual role of ZEB1 in the S100A8/A9-mediated metastasis in breast cancer cells that expressed ZEB1 at a significant level and showed that the ZEB1 was activated on the MCAM-downstream pathway upon S100A8/A9 binding. ZEB1 is well known to require Zn2+ for its activation based on the presence of several Zn-finger motifs in the transcription factor. However, how Zn2+-binding works on the pleiotropic role of ZEB1 through cancer progression has not been fully elucidated. We established the engineered cells, MDA-MB-231 MutZEB1 (MDA-MutZEB1), that stably express MutZEB1 (ΔZn). The cells were then evaluated in vitro for their invasion activities. Finally, an RNA-Seq analysis was performed to compare the gene alteration profiles of the established cells comprehensively. MDA-MutZEB1 showed a significant loss of the EMT, ultimately stalling the invasion. Inclusive analysis of the transcription changes after the expression of MutZEB1 (ΔZn) in MDA-MB-231 cells revealed the significant downregulation of LOX family genes, which are known to play a critical role in cancer metastasis. We found that LOXL1 and LOXL4 remarkably enhanced cancer invasiveness among the LOX family genes with altered expression. These findings indicate that ZEB1 potentiates Zn2+-mediated transcription of plural EMT-relevant factors, including LOXL1 and LOXL4, whose upregulation plays a critical role in the invasive dissemination of breast cancer cells.

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.

Production and optimization of a vasostatin-30 and vasoinhibin fusion protein that inhibits tumor angiogenesis and dissemination of breast cancer cells in a zebrafish model

Angiogenesis plays a key role in tumor growth and metastasis. Current antiangiogenic drugs have shown poor clinical benefit making the development of new antiangiogenic agents a clinical need. In this work, we optimized the production of a recombinant fusion protein derived from vasostatin-30 (VS30) and vasoinhibin (Vi), named VS_VI, using Escherichia coli and the central composite experimental design. The bioactivity of soluble VS_VI was compared to VS30, Vi, and angiostatin using in vitro cell cultures and the zebrafish model. Results showed that 21.3 and 17.5 mg/L of total VS_VI and soluble VS_VI, respectively, were attained at pH 7.0 and 28.5 °C. In vitro studies showed that purified soluble VS_VI decreased by 2-fold the VEGF-induced HUVEC proliferation compared to VS30 and Vi, showing no effect on 3T3 NIH cell proliferation at 10 nM. In vivo using zebrafish, VS_VI significantly reduced intratumoral angiogenesis at 1 mg/kg compared to a control, VS30, Vi, and angiostatin. VS_VI decreased metastatic dissemination of MDA-MB-468 and MDA-MB-231 cells at 1 mg/kg. Meanwhile, vasoinhibin and angiostatin at 1 mg/kg and vasostatin-30 at 10 mg/kg decreased the dissemination of T47D cells. Results showed VS_VI fusion protein impairs tumor angiogenesis and early dissemination of metastatic breast cancer cells, making it a promising agent in breast cancer treatment.

NR2F1 Is a Barrier to Dissemination of Early-Stage Breast Cancer Cells.

During early stages of breast cancer progression, HER2-mediated suppression of NR2F1 promotes dissemination by inducing EMT and a hybrid luminal/basal-like program.

Use of piggyBac Transposon System Constructed Murine Breast Cancer Model for Reporter Gene Imaging and Characterization of Metastatic Tumor Cells

PurposeThe piggyBac transposon system is known to non-viral integrate exogenous genes in the chromosomes of mammalian cells. This study exploited the reporter gene imaging, mediated by the piggyBac transposon system, to track the growth and dissemination of 4T1 triple-negative murine breast cancer cells in vivo. Subsequently, the study conducted an ex vivo analysis of the metastatic cells expressing reporter genes.MethodsA PB-3R-puro construct harboring multiple cistrons was used for piggyBac-mediated reporter genes imaging. These cistrons include monomeric red fluorescence protein (mRFP), luciferase 2 (luc2), and herpes simplex virus type I—thymidine kinase (HSV1-tk). In addition, 4T1 cells stably transfected with PB-3R-puro construct were implanted to Balb/C mice to form synergistic tumors. Further, liver metastatic 4T1 tumor was visualized by bioluminescent imaging. It was also excised to analyze cancer stem cell-related characteristics, including sphere formation, drug resistance, CD44 expression, and tumorigenesis.ResultsThe piggyBac transposon system did not influence the proliferation rate, invasion and migration rate, and mammosphere formation ability of 4T1 cells. The liver metastatic cells, named 4T1-3R_L cells, exhibited cancer stem cells (CSC)-related characteristics compared to parental 4T1 cells. We also found that 4T1-3R_L cells exhibited stronger migration and invasive abilities by wound healing assay and in vitro invasion assay, respectively. The microarray assay showed that epithelial–mesenchymal transition (EMT)-promoting markers, including vimentin, N-cadherin, Twist1, and Snail, were up-regulated, and the anti-EMT marker E-cadherin was down-regulated in 4T1-3R_L cells.ConclusionCurrent data suggest that the piggyBac transposon system can engineer cancer cells for tacking and characterizing tumor development in vivo and ex vivo.

Deregulated molecules and pathways in the predisposition and dissemination of breast cancer cells to bone

BackgroundBone metastasis is the most common metastatic destination in advanced breast cancer, presenting a poor prognosis and clinical challenges in management. To date, the mechanism of bone metastasis in breast cancer remains largely unclear. MethodsDifferentially expressed genes in primary tumours that developed bone metastases were systematically analysed using both TCGA-BRCA and E-MTAB-4003 databases. Adaptive phenotype in the subsequent bone lesions was analysed in the GSE46161 database. A series of biomarkers including homing, immune escape, angiogenesis, and factors involved in both osteoblastogenesis and osteoclastogenesis were included to dissect the molecular events underlying bone metastasis in breast cancer. ResultsUpregulated expressions of GDF11 expression is positively correlated with colonization, osteoblastogenesis and osteoclastogenesis, whilst CD151 is positively associated with angiogenesis and immune escape. PAFAH1B2 expression is inversely correlated with the angiogenic process. Reduced YTHDF2 may facilitate cancer cell homing, osteoclastogenesis and immune escape in breast cancer. DPP9, FAS, ZNF519, RPP14 and FAU were evaluated for their potential involvement in for the homing to bone, escaping from immune surveillance, angiogenesis, osteoblastic activity and osteoclastic activity in the multi-step process of bone metastasis. ConclusionGDF11, CD151, PAFAH1B2 and YTHDF2 may play a pivotal role in the predisposition of metastasis to the bone from breast cancer, whilst DPP9, FAS, ZNF519, RPP14 and FAU may be actively involved in the adaptative colonisation of metastatic breast cancer cells in bone.

MiR-205-5p Downregulation and ZEB1 Upregulation Characterize the Disseminated Tumor Cells in Patients with Invasive Ductal Breast Cancer

Background: Dissemination of breast cancer (BC) cells through the hematogenous or lymphogenous vessels leads to metastatic disease in one-third of BC patients. Therefore, we investigated the new prognostic features for invasion and metastasis. Methods: We evaluated the expression of miRNAs and epithelial-to-mesenchymal transition (EMT) genes in relation to CDH1/E-cadherin changes in samples from 31 patients with invasive ductal BC including tumor centrum (TU-C), tumor invasive front (TU-IF), lymph node metastasis (LNM), and CD45-depleted blood (CD45-DB). Expression of miRNA and mRNA was quantified by RT-PCR arrays and associations with clinico-pathological characteristics were statistically evaluated by univariate and multivariate analysis. Results: We did not verify CDH1 regulating associations previously described in cell lines. However, we did detect extremely high ZEB1 expression in LNMs from patients with distant metastasis, but without regulation by miR-205-5p. Considering the ZEB1 functions, this overexpression indicates enhancement of metastatic potential of lymphogenously disseminated BC cells. In CD45-DB samples, downregulated miR-205-5p was found in those expressing epithelial and/or mesenchymal markers (CTC+) that could contribute to insusceptibility and survival of hematogenously disseminated BC cells mediated by increased expression of several targets including ZEB1. Conclusions: miR-205-5p and potentially ZEB1 gene are promising candidates for markers of metastatic potential in ductal BC.

Breast cancer metastasis: immune profiling of lymph nodes reveals exhaustion of effector T cells and immunosuppression.

Sentinel lymph nodes are the first nodes draining the lymph from a breast and could reveal early changes in the host immune system upon dissemination of breast cancer cells. To investigate this, we performed single‐cell immune profiling of lymph nodes with and without metastatic cells. Whereas no significant changes were observed for B‐cell and natural killer (NK)‐cell subsets, metastatic lymph nodes had a significantly increased frequency of CD8 T cells and a skewing toward an effector/memory phenotype of CD4 and CD8 T cells, suggesting an ongoing immune response. Additionally, metastatic lymph nodes had an increased frequency of TIGIT (T‐cell immunoreceptor with Ig and ITIM domains)‐positive T cells with suppressed TCR signaling compared with non‐metastatic nodes, indicating exhaustion of effector T cells, and an increased frequency of regulatory T cells (Tregs) with an activated phenotype. T‐cell alterations correlated with the percentage of metastatic tumor cells, reflecting the presence of metastatic tumor cells driving T effector cells toward exhaustion and promoting immunosuppression by recruitment or increased differentiation toward Tregs. These results show that immune suppression occurs already in early stages of tumor progression.

Abstract 3154: Host and neoplastic cell Axl inhibition impair prostate and breast cancer bone metastasis

Abstract Prostate and breast cancers are the most prevalent and the second leading causes of cancer-deaths for men and women in the United States. Cancer survival rates generally worsen significantly with advanced and disseminated disease with nearly 90% of all cancer deaths resulting from metastasis. For breast and prostate cancers, bone is the most common site of metastasis. A key signaling pathway involved in metastatic dissemination of tumor cells is the receptor tyrosine kinase Axl. Axl belongs to the Tyro3, Axl, and MerTK (TAM) subfamily, is expressed in a variety of tumor types, including breast and prostate, and is associated with poor prognosis, metastasis, and outcome. The goal of the present study was to characterize the role of Axl signaling in prostate and breast cancer cell dissemination to bone. This was accomplished using Axl knockdown neoplastic cells, a selective small molecule Axl inhibitor (BGB324), and osteoclast progenitor cells in vitro, as well as intracardiac injection of Axl knockdown tumor cells in vivo. The results showed that Axl inhibition significantly decreases tumor cell migration and invasion in vitro, and suppression of Axl signaling in osteoclast precursor cells reduces the formation of mature osteoclasts. In vivo, Axl knockdown in prostate and breast cancer cells significantly suppressed the formation of neoplastic bone lesions. Taken together our findings indicate that therapeutic targeting of Axl may impair tumor metastasis to the bones through neoplastic and host cell signaling axes. Citation Format: Mai Tanaka, Dietmar W. Siemann. Host and neoplastic cell Axl inhibition impair prostate and breast cancer bone metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3154.

Analysis of disseminated tumor cells (DTCs) in primary breast cancer patients with various molecular subtypes.

e13000 Background: Despite successful treatment of the primary tumor, recurrence occurs in about 30% of breast cancer patients. One reason might be hematogenous spread during early disease stages. Disseminated breast cancer cells (DTCs) preferentially migrate into the bone marrow (BM) at early stages of the disease. Due to low proliferation, DTCs are persistent against systemic chemotherapy and might cause metastatic relapse at a later stage. Methods: BM aspirates were collected from the anterior iliac crest of patients with primary mamma carcinoma during surgery. After density gradient centrifugation cell suspensions were transferred onto glass slides and subjected to immunocytochemical staining against pan-cytokeratin. DTCs were visualized in pink using alkaline phosphatase and short counterstaining with hematoxylin which colored the nuclei light blue. DTCs were semi-automatically detected and enumerated using the Aperio Versa microscope based scanning system with a rare events algorithm that was trained to identify DTC candidates according to color, shape, intensity and size. As a positive control with each run, we used reference slides with a mix of bone marrow cells and a defined number of HCT116 cells. Results: Between February 2019 and December 2020 BM aspirates from 158 primary breast cancer patients were collected. Per patient about 4 million BM cells were analyzed. DTC detection revealed a positivity rate of 29% (46 patients). Molecular subtype analysis of DTC positive patients showed that 37% of the primary tumors (17 patients) were luminal A and 37% (17 patients) luminal B. In 9% of the cases (4 patients), tumors were HER2 enriched and 15% (8 patients) were triple negative. DTC count indicated that the majority of luminal B patients had 11-20 DTCs whereas luminal A patients tended to have lower DTC quantities varying between 1 and 10 DTCs. Conclusions: DTCs may serve as independent prognostic markers. Follow-Up data might reveal whether DTC quantification and molecular subtypes at primary diagnosis can be used to stratify patients at elevated risk for recurrence.

Dysregulation of tumour microenvironment driven by circ-TPGS2/miR-7/TRAF6/NF-κB axis facilitates breast cancer cell motility

Tumour microenvironment (TME) is frequently remodelled and deregulated in cancer development and progression. The underlying mechanisms are complex, multifactorial and largely unknown. Circular RNA (circRNA) is an endogenous RNA with a covalently closed loop that plays critical roles in the pathological and physiological processes of the organism. Here, we identified a TME-associated circRNA, circ-TPGS2, which promoted breast cancer (BC) cell dissemination via altering TME. High circ-TPGS2 was observed in metastatic BC tissues and cell lines in comparison to respective normal controls, which was linked to poor overall and recurrence-free survival. Overexpression of circ-TPGS2 notably promoted cell migration, while silencing of circ-TPGS2 resulted in an opposite trend. Moreover, circ-TPGS2 increased pro-inflammatory chemokine production and evoked tumour-associated inflammation by recruiting neutrophils via autocrine and paracrine manners. In terms of mechanism, circ-TPGS2 acted as a sponge of miR-7 and elevated TRAF6, leading to p65 phosphorylation and nuclear translocation, ultimately activating NF-κB signalling. In addition, p65 was abundantly occupied on circ-TPGS2 promoter, activating circ-TPGS2 transcription, thus, forming a positive feedback loop that amplified the pro-metastasis effect of circ-TPGS2. Taken together, our data for the first time reveal the biological implication of circ-TPGS2 in BC, it triggers TME reshaping that facilitates BC metastasis through the miR-7/TRAF6/NF-κB signalling cascade.

Osteoblast-Derived Paracrine and Juxtacrine Signals Protect Disseminated Breast Cancer Cells from Stress.

Simple SummaryBone metastasis is a debilitating, incurable complication that occurs in breast cancer patients with a variable period of latency after treatment of the primary tumor. The seeding of individual cancer cells in the bone marrow from the primary tumor occurs early in disease progression. How disseminated cancer cells are able to remain dormant in the bone marrow and then reactivate decades later to form destructive bone lesions is not fully understood. We have used model systems to identify bone resident cell types, and molecular mediators, that influence the survival and fate of disseminated breast cancer cells. The identification of such cellular and molecular support mechanisms can inform the design of new therapeutic strategies aimed at eliminating indolent cancer cells from the bone marrow, preventing progression to overt metastases.Metastatic breast cancer in bone is incurable and there is an urgent need to develop new therapeutic approaches to improve survival. Key to this is understanding the mechanisms governing cancer cell survival and growth in bone, which involves interplay between malignant and accessory cell types. Here, we performed a cellular and molecular comparison of the bone microenvironment in mouse models representing either metastatic indolence or growth, to identify mechanisms regulating cancer cell survival and fate. In vivo, we show that regardless of their fate, breast cancer cells in bone occupy niches rich in osteoblastic cells. As the number of osteoblasts in bone declines, so does the ability to sustain large numbers of breast cancer cells and support metastatic outgrowth. In vitro, osteoblasts protected breast cancer cells from death induced by cell stress and signaling via gap junctions was found to provide important juxtacrine protective mechanisms between osteoblasts and both MDA-MB-231 (TNBC) and MCF7 (ER+) breast cancer cells. Combined with mathematical modelling, these findings indicate that the fate of DTCs is not controlled through the association with specific vessel subtypes. Instead, numbers of osteoblasts dictate availability of protective niches which breast cancer cells can colonize prior to stimulation of metastatic outgrowth.

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.