Polyclonal breast cancer metastases arise from collective dissemination of keratin 14-expressing tumor cell clusters

Recent genomic studies challenge the conventional model that each metastasis must arise from a single tumor cell and instead reveal that metastases can be composed of multiple genetically distinct clones. These intriguing observations raise the question: How do polyclonal metastases emerge from the primary tumor? In this study, we used multicolor lineage tracing to demonstrate that polyclonal seeding by cell clusters is a frequent mechanism in a common mouse model of breast cancer, accounting for >90% of metastases. We directly observed multicolored tumor cell clusters across major stages of metastasis, including collective invasion, local dissemination, intravascular emboli, circulating tumor cell clusters, and micrometastases. Experimentally aggregating tumor cells into clusters induced a >15-fold increase in colony formation ex vivo and a >100-fold increase in metastasis formation in vivo. Intriguingly, locally disseminated clusters, circulating tumor cell clusters, and lung micrometastases frequently expressed the epithelial cytoskeletal protein, keratin 14 (K14). RNA-seq analysis revealed that K14⁺ cells were enriched for desmosome and hemidesmosome adhesion complex genes, and were depleted for MHC class II genes. Depletion of K14 expression abrogated distant metastases and disrupted expression of multiple metastasis effectors, including Tenascin C (Tnc), Jagged1 (Jag1), and Epiregulin (Ereg). Taken together, our findings reveal K14 as a key regulator of metastasis and establish the concept that K14⁺ epithelial tumor cell clusters disseminate collectively to colonize distant organs.During metastasis, cancer cells escape the primary tumor, travel through the circulation, and colonize distant organs. Conventional models of cancer progression propose that each metastasis arises from the clonal outgrowth of a single tumor cell and this conceptual framework is a foundation for models, such as epithelial-mesenchymal transition (EMT) and migratory cancer stem cells (1).Challenging the generality of the single-cell/single-metastasis model are long-standing clinical observations that tumor cell clusters (also termed “tumor clumps”) are also observed across the stages of metastasis. Tumor cell clusters are detected in the bloodstream of cancer patients (2), clusters can efficiently seed metastases (3), and though rare, circulating tumor cell (CTC) clusters have prognostic significance (4, 5). Furthermore, metastases are composed of multiple genetically distinct tumor cell clones, in mouse models of breast, pancreas, and small cell carcinoma (5–7), and in human metastatic prostate cancer patients (8). Taken together, these observations provide accumulating evidence that tumor cell clusters contribute to metastasis. However, they leave unresolved two important questions: how do tumor cell clusters emerge from the primary tumor, and which molecular features identify cell clusters that metastasize?An important clinical observation is that cancer cells invade the surrounding stroma as cohesive clusters in the majority of epithelial tumors, a process termed “collective invasion” (9, 10). In breast cancer, collective invasion is facilitated by invasive leader cells, a subpopulation of tumor cells that highly express keratin 14 (K14) and other basal epithelial markers (11). K14⁺ cells are migratory, protrusive, and lead trailing K14⁻ cells, while maintaining cell–cell cohesion and E-cadherin–based cell contacts.In this study, we sought to understand how these K14⁺ cells exit collective invasion strands in the primary tumor and travel to distant organs (12). One hypothesis is that collective invasion is an intermediate step toward eventual single-cell dissemination and monoclonal metastasis. However, tumor cell clusters are detected in circulation (5) and primary human breast tumors can disseminate collectively into the surrounding extracellular matrix in ex vivo assays (13–15). These data prompted an alternative hypothesis, that collectively invading K14⁺ cancer cells could initiate and complete the metastatic process as a cohesive multicellular unit. Here we define the clonal nature of metastases in a spontaneous mouse model of metastasis to the lungs (16, 17), in which the predominant invasive form in the primary tumor is collective invasion strands led by K14⁺ cells (11). We establish that the majority of metastases arise from polyclonal seeds, and show that disseminated tumor cell clusters are predominantly composed of K14⁺ cells. We propose a mechanism for polyclonal metastasis via the collective invasion, dissemination, and colonization of clusters of K14⁺ cancer cells.     

Role of the B1 kinin receptor in the regulation of cardiac function and remodeling after myocardial infarction

Kinins exert cardioprotective effects via 2 G-protein-coupled receptors, B1 and B2. Using B1 kinin receptor gene knockout mice (B1-/-), we tested the hypotheses that the B1 receptor plays an important role in preservation of cardiac function, whereas lack of B1 may accelerate cardiac remodeling and dysfunction after myocardial infarction, and that B2 receptors may compensate for lack of B1, whereas blockade of B2 receptors in B1-/- mice may cause further deterioration of cardiac function and remodeling. Female B1-/- mice and wild-type controls (C57BL/6J, B1+/+) underwent sham surgery or myocardial infarction and were treated with either vehicle or B2-antagonist (icatibant, 500 microg/kg per day, subcutaneous) for 8 weeks. We found that in sham myocardial infarction, B1-/- mice had a larger left ventricular diastolic chamber dimension both initially and at 4 to 8 weeks compared with B1+/+. Left ventricular mass and myocyte size were also larger in B1-/- with sham operation than in B1+/+, although cardiac function did not differ between strains. After myocardial infarction, cardiac remodeling and function were similar in both strains, although B1-/- mice tended to have lower blood pressure. Blockade of B2 receptors tended to worsen cardiac remodeling and dysfunction in B1-/- but not in B1+/+. These results may suggest that B2 receptors play an important role in compensating for lack of B1 receptors in mice with myocardial infarction. Dual blockade of both B1 and B2 eliminates this compensation, leading to further deterioration of cardiac dysfunction and remodeling after myocardial infarction.     

Environmental Isolates of Azole-Resistant Aspergillus fumigatus in Germany

Azole antifungal drug resistance in Aspergillus fumigatus is an emerging problem in several parts of the world. Here we investigated the distribution of such strains in soils from Germany. At a general positivity rate of 12%, most prevalently, we found strains with the TR₃₄/L98H and TR₄₆/Y121F/T289A alleles, dispersed along a corridor across northern Germany. Comparison of the distributions of resistance alleles and genotypes between environment and clinical samples suggests the presence of local clinical clusters.     

A new method to evaluate the effects of shear on the skin

Currently, pressure ulcer preventive strategies focus mainly on pressure redistribution. Little attention is paid to reduce the harmful effects of shear‐force, because little is known about pathophysiological aspects of shear‐force. Even today, no method to measure the effects of shear‐force on the skin is available. Therefore, the aim of this study was to investigate the response to shear‐forces in terms of analyzing a noninvasive biomarker and reactive hyperemic parameter measured at the skin of healthy participants. A physical model was developed to produce a combination of pressure and shear or pressure alone on the skin. Ten healthy male participants were included and pressure (3.9 kPa) and a combined loading of pressure and shear (2.4 kPa + 14.5 N) was applied at the volar aspect of the forearms for 15 and 30 minutes. A Sebutape sample was used to collect IL‐1α and total protein (TP) noninvasively. The reactive hyperemic parameter was derived from a laser Doppler flowmeter. The increase in IL‐1α/TP‐ratio after a combined loading of pressure and shear for 30 minutes of 6.2 ± 2.5 was significantly higher compared with all other test conditions (p < 0.05). The increase in cutaneous blood cell flux was already significantly higher when a combined loading of pressure and shear was applied for 15 minutes compared with pressure alone. These results shows that the IL‐1α/TP‐ratio and cutaneous blood cell flux can be used as robust measures of the effect of shear‐force on skin in humans. Therefore, this model can be used to evaluate materials aimed at the reduction of shear.     

Influence of <Emphasis Type="Italic">MX1</Emphasis> promoter rs2071430 G/T polymorphism on susceptibility to systemic lupus erythematosus

The expression of interferon inducible genes are reported to be heightened in systemic lupus erythematosus (SLE); nevertheless, not much is known regarding the genetic variants underlying these genes and their role in the pathogenesis of disease. Herein, we aim to explore the potential association and contribution of polymorphisms in MX1 gene (i) promoter with part of exon 1 (ii) intron 6, and (iii) their resulting haplotypes, with susceptibility to SLE. A total of 306 subjects, 152 SLE and 154 healthy controls (HC), were screened by direct sequencing method. Statistical analysis was carried out using appropriate software. The screening region of interest in MX1 revealed the existence of promoter (?123C/A, ?88G/T, ?20 A/C) and intron 6 (+9862G/A, +10190G/A, +9901C/G, +9920C/A, +9959C/T, +10047A/G) variants in SLE and HC. A significant association was observed between MX1 ?88G/T SNP and susceptibility to SLE (χ ²?=?4.18, p?=?0.04, OR?=?1.89, 95 % CI 1.03–3.5). Haplotype analysis also revealed increased risk of SLE among individuals carrying CTA haplotype (?123 C, ?88 T, ?20 A) (χ ²?=?5.74, p?=?0.017, OR?=?4.28, 95 % CI 1.30–14.06). None of the other tested variants showed any significant association with SLE. The present study is the first to reveal the influence of genetic variation in MX1 gene in susceptibility to SLE.     

Risk indicators for the presence and extent of root caries among caries-active adults enrolled in the Xylitol for Adult Caries Trial (X-ACT)

Objective

This paper uses baseline data from a randomized clinical trial to evaluate cross-sectional indicators of root caries in caries-active adults.

Materials and methods

Adults (21–80?years) having at least 12 erupted teeth and between one and ten caries lesions were enrolled. Participants (n?=?437) received caries exams by trained, calibrated examiners and responded to baseline demographic and medical–dental questionnaires. We examined associations between baseline characteristics and (1) the presence of any root caries using Mantel–Haenszel hypothesis tests and odds ratio (OR) estimators and (2) the number of root surfaces with caries among study participants with exposed root surfaces (n?=?349) using Mantel–Haenszel mean score tests and Mann–Whitney estimators.

Results/conclusions

Adjusting for study site and age, male gender [OR, 1.72; 95% confidence interval (CI), 1.08, 2.78], white race (OR, 2.39; 95% CI, 1.43, 3.98), recent dental visit (OR, 1.98; 95% CI, 1.07, 3.66), poor self-described oral health (OR, 2.65; 95% CI, 1.10, 6.39), and recent professional fluoride treatment (OR, 1.85; 95% CI, 1.06, 3.25) were significantly associated with increased odds to have any root caries, and study participants with exposed root surfaces characterized by male gender [Mann–Whitney probability estimate (MW)?=?0.57; 95% CI, 0.51, 0.63), white race (MW, 0.61; 0.55, 0.68), recent dental visit (MW, 0.58; 0.50, 0.67), poor self-described oral health (MW, 0.61; 0.53, 0.69), and flossing at least once per day (MW, 0.57; 95% CI, 0.51, 0.62) were significantly more likely to have a greater number of root surfaces with caries than a randomly selected study participant from their respective complementary subgroups (female gender, non-white, etc.).

Clinical relevance

Our findings may help identify individuals at higher root caries risk.     

Transconjunctival versus subciliary approach for orbital fracture repair—an anthropometric evaluation of 221 cases

Objectives

In the literature, there is an ongoing discussion about the influence of orbital fractures and the surgical approach on the rate of eyelid deformities of the lower eyelid.

Materials and methods

We present an evaluation of a series of 221 patients 9 months after zygomaticomaxillary complex fracture repair that underwent implant removal. Reference anthropometric data were measured on standardized pre- and postoperative photographs. Analysis included eye fissure width and height, lid sulcus and upper lid height, upper and lower iris coverage, position of cornea to palpebra inferior, canthal tilt, scleral show, ectropion, and entropion. Both operated and contralateral eyelids were evaluated as well as whether a transconjunctival or a subciliary approach was performed.

Results

Time, surgery, and surgical approach presented significant effects on eye fissure index and lower iris coverage. Scleral show was significantly influenced by the surgical procedure itself as well as by the type of incision. The rate of ectropion increased significantly pre- to postoperative.

Conclusions

The subciliary approach included the highest risk of lower lid retraction. The low pre- to postoperative increase of scleral show and ectropion compared to recent studies gives us an idea about the influence of the underlying trauma on the rate of lower lid retraction. The standardized measurements described are accurately and objective to evaluate postoperative results.

Clinical relevance

The transconjunctival approach is preferable in orbital fracture repair.