Abstract
Circulating tumor cell (CTC) clusters mediate metastasis at a higher efficiency and are associated with lower overall survival in breast cancer compared to single cells. Combining ...single-cell RNA sequencing and protein analyses, here we report the profiles of primary tumor cells and lung metastases of triple-negative breast cancer (TNBC). ICAM1 expression increases by 200-fold in the lung metastases of three TNBC patient-derived xenografts (PDXs). Depletion of ICAM1 abrogates lung colonization of TNBC cells by inhibiting homotypic tumor cell-tumor cell cluster formation. Machine learning-based algorithms and mutagenesis analyses identify ICAM1 regions responsible for homophilic ICAM1-ICAM1 interactions, thereby directing homotypic tumor cell clustering, as well as heterotypic tumor-endothelial adhesion for trans-endothelial migration. Moreover, ICAM1 promotes metastasis by activating cellular pathways related to cell cycle and stemness. Finally, blocking ICAM1 interactions significantly inhibits CTC cluster formation, tumor cell transendothelial migration, and lung metastasis. Therefore, ICAM1 can serve as a novel therapeutic target for metastasis initiation of TNBC.
Circulating tumor cells (CTC) seed cancer metastases; however, the underlying cellular and molecular mechanisms remain unclear. CTC clusters were less frequently detected but more metastatic than ...single CTCs of patients with triple-negative breast cancer and representative patient-derived xenograft models. Using intravital multiphoton microscopic imaging, we found that clustered tumor cells in migration and circulation resulted from aggregation of individual tumor cells rather than collective migration and cohesive shedding. Aggregated tumor cells exhibited enriched expression of the breast cancer stem cell marker CD44 and promoted tumorigenesis and polyclonal metastasis. Depletion of CD44 effectively prevented tumor cell aggregation and decreased PAK2 levels. The intercellular CD44-CD44 homophilic interactions directed multicellular aggregation, requiring its N-terminal domain, and initiated CD44-PAK2 interactions for further activation of FAK signaling. Our studies highlight that CD44
CTC clusters, whose presence is correlated with a poor prognosis of patients with breast cancer, can serve as novel therapeutic targets of polyclonal metastasis. SIGNIFICANCE: CTCs not only serve as important biomarkers for liquid biopsies, but also mediate devastating metastases. CD44 homophilic interactions and subsequent CD44-PAK2 interactions mediate tumor cluster aggregation. This will lead to innovative biomarker applications to predict prognosis, facilitate development of new targeting strategies to block polyclonal metastasis, and improve clinical outcomes.
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Tumor-initiating cells with reprogramming plasticity or stem-progenitor cell properties (stemness) are thought to be essential for cancer development and metastatic regeneration in many cancers; ...however, elucidation of the underlying molecular network and pathways remains demanding. Combining machine learning and experimental investigation, here we report CD81, a tetraspanin transmembrane protein known to be enriched in extracellular vesicles (EVs), as a newly identified driver of breast cancer stemness and metastasis. Using protein structure modeling and interface prediction-guided mutagenesis, we demonstrate that membrane CD81 interacts with CD44 through their extracellular regions in promoting tumor cell cluster formation and lung metastasis of triple negative breast cancer (TNBC) in human and mouse models. In-depth global and phosphoproteomic analyses of tumor cells deficient with CD81 or CD44 unveils endocytosis-related pathway alterations, leading to further identification of a quality-keeping role of CD44 and CD81 in EV secretion as well as in EV-associated stemness-promoting function. CD81 is coexpressed along with CD44 in human circulating tumor cells (CTCs) and enriched in clustered CTCs that promote cancer stemness and metastasis, supporting the clinical significance of CD81 in association with patient outcomes. Our study highlights machine learning as a powerful tool in facilitating the molecular understanding of new molecular targets in regulating stemness and metastasis of TNBC.
Background
Clinical and genomic data from patients with early-stage breast cancer suggest more aggressive disease in premenopausal women. However, the association between age, disease course, and ...molecular profile from liquid biopsy in metastatic breast cancer (MBC) is not well characterized.
Methods
Patients were classified as premenopausal (< 45 years), perimenopausal (45–55 years), or postmenopausal (> 55 years). Cohort 1 consisted of patients with MBC who consented for prospective serial evaluation of circulating tumor cells (CTCs) using CellSearch™. Cohort 2 included patients who, as part of routine care, had circulating tumor DNA (ctDNA) sequenced by the Guardant360™ assay. Clinicopathologic data were collected from retrospective review to compare disease features between premenopausal and postmenopausal women.
Results
Premenopausal women represented 26% of 138 patients in Cohort 1 and 21% of 253 patients in Cohort 2. In Cohort 1, younger patients had a shorter time to metastases and a higher prevalence of lung and brain metastases. Overall, there were similar rates of ≥ 5 CTCs/7.5 mL, HER2 + CTC expression, and CTC clusters between pre- and postmenopausal women. However, for those with triple negative breast cancer, premenopausal women had a higher proportion of ≥ 5 CTCs/7.5 mL. In Cohort 2, premenopausal women had a higher incidence of
FGFR1
(OR 2.75,
p
= 0.022) and
CCND2
(OR 6.91,
p
= 0.024) alterations. There was no difference in the ctDNA mutant allele frequency or the number of detected alterations between these age groups.
Conclusions
Our data reveal that premenopausal women diagnosed with MBC have unique clinical, pathologic, and molecular features when compared to their postmenopausal counterparts. Our results highlight
FGFR1
inhibitors as potential therapeutics of particular interest among premenopausal women.
Bone and dentin development requires temporal and spatial deposition of calcium phosphate mineral. A host of proteins works in concert to contribute to this tightly regulated process while ...malfunction in this scheme often leads to pathological defects. We have reported earlier that DMP1 stimulation of preosteoblasts leads to calcium release from internal Ca2+ stores and this store depletion is sensed by the ER Ca2+ sensor STIM1 (stromal interaction molecule 1). In this study, we first assessed the temporal and spatial localization of STIM1 protein during the development of bone and dentin by immunohistochemical methods. We further analyzed the function of STIM1 by establishing a stable MC3T3-E1 cell-line by overexpressing STIM1 (MC3T3-E1/STIM1 OE). Under mineralizing conditions, STIM1 overexpressing cells showed increased calcium deposits with higher expression of key osteogenic markers, such as Runx2 and type I collagen, BMP4 when compared with the control cells. Our results demonstrate that during mineralized matrix formation STIM1, the key ER sensor protein, can promote cellular differentiation in the presence of extracellular calcium.
Celotno besedilo
Dostopno za:
BFBNIB, DOBA, GIS, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Purpose
Meaningful comparison of mutational landscapes across ethnic groups requires the use of standardized platform technology. We have used a harmonized NGS-based liquid biopsy assay to explore ...the differential genomic landscape of patients with initially hormone receptor-positive (HR+), HER2-negative MBC of first line metastasis or primary Stage IV at diagnosis from the United States (US) and China (CN).
Methods
Plasma circulating tumor DNA (ctDNA) from 27 US patients and 65 CN patients was sequenced using the harmonized CLIA-certified, 152-gene PredicineCare™ liquid biopsy assay. Kaplan–Meier survival analysis was performed to analyze the correlation between genomic alterations and progression-free survival (PFS), and
p
-values were calculated using the log-rank test.
Results
All patients in the CN cohort received chemotherapy and/or hormonal therapy, while 85.2% (23/27) patients in the US cohort received hormonal therapy plus CDK4/6 inhibitors. Mutations were detected in 23 of 27 (85%) US patients and 54 of 65 (83%) CN patients. The prevalence of
AKT1
(
P
= 0.008) and
CDH1
(
P
= 0.021) alterations were both higher in the US vs. CN cohort. In addition,
FGFR1
amplification were more frequent in the CN vs. US cohort (
P
= 0.048).
PTEN
deletions (
P
= 0.03) and
ESR1
alterations (
P
= 0.02) were associated with shorter PFS in the CN cohort, neither of these associations were observed in the US cohort. Interestingly, a reduced association between
PTEN
deletion and PFS was observed in patients receiving CDK4/6 inhibitor treatment.
Conclusion
The differential prevalence of ctDNA-based alterations such as
FGFR1
,
AKT1
, and
CDH1
was observed in initially HR+/HER2− MBC patients in the US vs. CN. In addition, the association of
PTEN
deletions with shorter PFS was found in the CN but not the US cohort. The differential genomic landscapes across the two ethnic groups may reflect biologic differences and clinical implications.
Accumulating evidence demonstrates that circulating tumor cell (CTC) clusters have higher metastatic ability than single CTCs and negatively correlate with cancer patient outcomes. Along with ...homotypic CTC clusters, heterotypic CTC clusters (such as neutrophil-CTC clusters), which have been identified in both cancer mouse models and cancer patients, lead to more efficient metastasis formation and worse patient outcomes. However, the mechanism by which neutrophils bind to CTCs remains elusive. In this study, we found that intercellular adhesion molecule-1 (ICAM-1) on triple-negative breast cancer (TNBC) cells and CD11b on neutrophils mediate tumor cell-neutrophil binding. Consequently, CD11b deficiency inhibited tumor cell-neutrophil binding and TNBC metastasis. Furthermore, CD11b mediated hydrogen peroxide (H
O
) production from neutrophils. Moreover, we found that ICAM-1 in TNBC cells promotes tumor cells to secrete suPAR, which functions as a chemoattractant for neutrophils. Knockdown of uPAR in ICAM-1
TNBC cells reduced lung-infiltrating neutrophils and lung metastasis. Bioinformatics analysis confirmed that uPAR is highly expressed in TNBCs, which positively correlates with higher neutrophil infiltration and negatively correlates with breast cancer patient survival. Collectively, our findings provide new insight into how neutrophils bind to CTC to facilitate metastasis and discover a novel potential therapeutic strategy by blocking the ICAM-1-suPAR-CD11b axis to inhibit TNBC metastasis.
The clinical relevance of circulating tumor cell clusters (CTC-clusters) in breast cancer (BC) has been mostly studied using the CellSearch®, a marker-dependent method detecting only ...epithelial-enriched clusters. However, due to epithelial-to-mesenchymal transition, resorting to marker-independent approaches can improve CTC-cluster detection. Blood samples collected from healthy donors and spiked-in with tumor mammospheres, or from BC patients, were processed for CTC-cluster detection with 3 technologies: CellSearch®, CellSieve™ filters, and ScreenCell® filters. In spiked-in samples, the 3 technologies showed similar recovery capability, whereas, in 19 clinical samples processed in parallel with CellSearch® and CellSieve™ filters, filtration allowed us to detect more CTC-clusters than CellSearch® (median number = 7 versus 1, p = 0.0038). Next, samples from 37 early BC (EBC) and 23 metastatic BC (MBC) patients were processed using ScreenCell® filters for attaining both unbiased enrichment and marker-independent identification (based on cytomorphological criteria). At baseline, CTC-clusters were detected in 70% of EBC cases and in 20% of MBC patients (median number = 2, range 0–20, versus 0, range 0–15, p = 0.0015). Marker-independent approaches for CTC-cluster assessment improve detection and show that CTC-clusters are more frequent in EBC than in MBC patients, a novel finding suggesting that dissemination of CTC-clusters is an early event in BC natural history.
Circulating tumor cells (CTCs) captured from the blood of cancer patients may serve as a surrogate source of tumor material that can be obtained via a venipuncture (also known as a liquid biopsy) and ...used to better understand tumor characteristics. However, the only FDA-cleared CTC assay has been limited to the enumeration of surface marker–defined cells and not further characterization of the CTCs. In this study, we tested the ability of a semi-automated device capable of capturing and harvesting CTCs from peripheral blood based on cell size and deformability, agnostic of cell-surface markers (the Parsortix® PC1 System), to yield CTCs for evaluation by downstream techniques commonly available in clinical laboratories. The data generated from this study were used to support a De Novo request (DEN200062) for the classification of this device, which the FDA recently granted. As part of a multicenter clinical trial, peripheral blood samples from 216 patients with metastatic breast cancer (MBC) and 205 healthy volunteers were subjected to CTC enrichment. A board-certified pathologist enumerated the CTCs from each participant by cytologic evaluation of Wright-Giemsa-stained slides. As proof of principle, cells harvested from a concurrent parallel sample provided by each participant were evaluated using one of three additional evaluation techniques: molecular profiling by qRT-PCR, RNA sequencing, or cytogenetic analysis of HER2 amplification by FISH. The study demonstrated that the Parsortix® PC1 System can effectively capture and harvest CTCs from the peripheral blood of MBC patients and that the harvested cells can be evaluated using orthogonal methodologies such as gene expression and/or Fluorescence In Situ Hybridization (FISH).
Tooth enamel develops within a pH sensitive amelogenin-rich protein matrix. The purpose of the present study is to shed light on the intimate relationship between enamel matrix pH, enamel protein ...self-assembly, and enamel crystal growth during early amelogenesis. Universal indicator dye staining revealed highly acidic pH values (pH 3-4) at the exocytosis site of secretory ameloblasts. When increasing the pH of an amelogenin solution from pH 5 to pH 7, there was a gradual increase in subunit compartment size from 2 nm diameter subunits at pH 5 to a stretched configuration at pH6 and to 20 nm subunits at pH 7. HSQC NMR spectra revealed that the formation of the insoluble amelogenin self-assembly structure at pH6 was critically mediated by at least seven of the 11 histidine residues of the amelogenin coil domain (AA 46-117). Comparing calcium crystal growth on polystyrene plates, crystal length was more than 20-fold elevated at pH 4 when compared to crystals grown at pH 6 or pH 7. To illustrate the effect of pH on enamel protein self-assembly at the site of initial enamel formation, molar teeth were immersed in phosphate buffer at pH4 and pH7, resulting in the formation of intricate berry tree-like assemblies surrounding initial enamel crystal assemblies at pH4 that were not evident at pH7 nor in citrate buffer. Amelogenin and ameloblastin enamel proteins interacted at the secretory ameloblast pole and in the initial enamel layer, and co-immunoprecipitation studies revealed that this amelogenin/ameloblastin interaction preferentially takes place at pH 4-pH 4.5. Together, these studies highlight the highly acidic pH of the very early enamel matrix as an essential contributing factor for enamel protein structure and self-assembly, apatite crystal growth, and enamel protein interactions.
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