Red Blood Cells Protein Profile Is Modified in Breast Cancer Patients.

Metastasis is the primary cause of death for most breast cancer (BC) patients who succumb to the disease. During the hematogenous dissemination, circulating tumor cells interact with different blood components. Thus, there are microenvironmental and systemic processes contributing to cancer regulation. We have recently published that red blood cells (RBCs) that accompany circulating tumor cells have prognostic value in metastatic BC patients. RBC alterations are related to several diseases. Although the principal known role is gas transport, it has been recently assigned additional functions as regulatory cells on circulation. Hence, to explore their potential contribution to tumor progression, we characterized the proteomic composition of RBCs from 53 BC patients from stages I to III and IV, compared with 33 cancer-free controls. In this work, we observed that RBCs from BC patients showed a different proteomic profile compared to cancer-free controls and between different tumor stages. The differential proteins were mainly related to extracellular components, proteasome, and metabolism. Embryonic hemoglobins, not expected in adults' RBCs, were detected in BC patients. Besides, lysosome-associated membrane glycoprotein 2 emerge as a new RBCs marker with diagnostic and prognostic potential for metastatic BC patients. Seemingly, RBCs are acquiring modifications in their proteomic composition that probably represents the systemic cancer disease, conditioned by the tumor microenvironment.

Methodology for the Isolation and Analysis of CTCs.

The majority of deaths related to breast cancer are caused by metastasis. Understanding the process of metastasis is key to achieve a reduction on breast cancer mortality. Currently, liquid biopsies are gaining attention in this regard. Circulating tumor cells (CTCs), an important component of liquid biopsies, are cells shed from primary tumor that disseminate to blood circulation being responsible of distal metastasis. Hence, the study CTCs is a promising alternative to monitor the progress of metastasis disease and can be used for early diagnosis of cancers as well as for earlier assessment of cancer recurrence and therapy efficacy. Despite their clinical interest, CTC analysis is not recommended by oncology guidelines so far. The main reason is that there is no gold standard technology for CTCs isolation and most of the current technologies are not yet validated for clinical use. In this chapter we will focus on the most relevant technologies for CTC isolation based on their properties and depending on whether it is a positive or negative selection. We also describe each technology based on its potential use and its relevance in breast cancer. The chapter also contains a future perspective including the challenges and requirements of CTC detection.

CTCs-derived xenograft development in a triple negative breast cancer case.

Triple-negative breast cancer (TNBC) is characterized by high rates of metastasis and no available molecular targets. CTCs derived xenografts (CDX) have demonstrated to be a promising tool for understanding cancer biology. In our study, a CDX from a TNBC patient was developed for the first time. After CDX characterization, WNT signaling was found as the main mechanism related with this tumor biology and potential CTCs markers were identified and subsequently validated in TNBC patients. In this cohort high levels of MELK expression were associated with poorer survival rates. Overall, our study demonstrates that CTCs from TNBC are tumorigenic and CDXs are a useful model to obtain valuable information about the tumor.

The Rho exchange factors Vav2 and Vav3 favor skin tumor initiation and promotion by engaging extracellular signaling loops.

The catalytic activity of GDP/GTP exchange factors (GEFs) is considered critical to maintain the typically high activity of Rho GTPases found in cancer cells. However, the large number of them has made it difficult to pinpoint those playing proactive, nonredundant roles in tumors. In this work, we have investigated whether GEFs of the Vav subfamily exert such specific roles in skin cancer. Using genetically engineered mice, we show here that Vav2 and Vav3 favor cooperatively the initiation and promotion phases of skin tumors. Transcriptomal profiling and signaling experiments indicate such function is linked to the engagement of, and subsequent participation in, keratinocyte-based autocrine/paracrine programs that promote epidermal proliferation and recruitment of pro-inflammatory cells. This is a pathology-restricted mechanism because the loss of Vav proteins does not cause alterations in epidermal homeostasis. These results reveal a previously unknown Rho GEF-dependent pro-tumorigenic mechanism that influences the biology of cancer cells and their microenvironment. They also suggest that anti-Vav therapies may be of potential interest in skin tumor prevention and/or treatment.

Biosensors for the detection of circulating tumour cells.

Metastasis is the cause of most cancer deaths. Circulating tumour cells (CTCs) are cells released from the primary tumour into the bloodstream that are considered the main promoters of metastasis. Therefore, these cells are targets for understanding tumour biology and improving clinical management of the disease. Several techniques have emerged in recent years to isolate, detect, and characterise CTCs. As CTCs are a rare event, their study requires multidisciplinary considerations of both biological and physical properties. In addition, as isolation of viable cells may give further insights into metastatic development, cell recovery must be done with minimal cell damage. The ideal system for CTCs analysis must include maximum efficiency of detection in real time. In this sense, new approaches used to enrich CTCs from clinical samples have provided an important improvement in cell recovery. However, this progress should be accompanied by more efficient strategies of cell quantification. A range of biosensor platforms are being introduced into the technology for CTCs quantification with promising results. This review provides an update on recent progress in CTCs identification using different approaches based on sensor signaling.

Isolation of Single Circulating Tumor Cells Using VyCAP Puncher System.

Tumor heterogeneity has a major role in the development of tumor evasion and resistance to treatments. To study and understand the intrinsic heterogeneity of cancer cells, the use of single-cell isolation technology has had a major boost in recent years, gaining ground to bulk analysis in the study of solid tumors. In the liquid biopsy field, the use of technologies for single-cell analysis has represented a major advance in the study of the heterogeneity of circulating tumor cells (CTCs), providing relevant information about therapy-resistant CTCs. However, single-cell analysis of CTCs is still challenging due to the weakness and scarcity of these cells. In this chapter, we describe a protocol for CTCs isolation at a single-cell level using the VyCAP Puncher system.

The thyroid hormone receptors as modulators of skin proliferation and inflammation.

We have analyzed the role of the thyroid hormone receptors (TRs) in epidermal homeostasis. Reduced keratinocyte proliferation is found in interfollicular epidermis of mice lacking the thyroid hormone binding isoforms TRα1 and TRß (KO mice). Similar results were obtained in hypothyroid animals, showing the important role of the liganded TRs in epidermal proliferation. In addition, KO and hypothyroid animals display decreased hyperplasia in response to 12-O-tetradecanolyphorbol-13-acetate. Both receptor isoforms play overlapping functional roles in the skin because mice lacking individually TRα1 or TRß also present a proliferative defect but not as marked as that found in double KO mice. Defective proliferation in KO mice is associated with reduction of cyclin D1 expression and up-regulation of the cyclin-dependent kinase inhibitors p19 and p27. Paradoxically, ERK and AKT activity and expression of downstream targets, such as AP-1 components, are increased in KO mice. Increased p65/NF-κB and STAT3 phosphorylation and, as a consequence, augmented expression of chemokines and proinflammatory cytokines is also found in these animals. These results show that thyroid hormones and their receptors are important mediators of skin proliferation and demonstrate that TRs act as endogenous inhibitors of skin inflammation, most likely due to interference with AP-1, NF-κB, and STAT3 activation.

A genome-wide cell-free DNA methylation analysis identifies an episignature associated with metastatic luminal B breast cancer.

Breast cancers of the luminal B subtype are frequent tumors with high proliferation and poor prognosis. Epigenetic alterations have been found in breast tumors and in biological fluids. We aimed to profile the cell-free DNA (cfDNA) methylome of metastatic luminal B breast cancer (LBBC) patients using an epigenomic approach to discover potential noninvasive biomarkers. Plasma cfDNA was analyzed using the Infinium MethylationEpic array in a cohort of 14 women, including metastatic LBBC patients and nontumor controls. The methylation levels of cfDNA and tissue samples were validated with droplet digital PCR. The methylation and gene expression data of 582 primary luminal breast tumors and 79 nontumor tissues were obtained from The Cancer Genome Atlas (TCGA). We found an episignature of 1,467 differentially methylated CpGs that clearly identified patients with LBBC. Among the genes identified, the promoter hypermethylation of WNT1 was validated in cfDNA, showing an area under the ROC curve (AUC) of 0.86 for the noninvasive detection of metastatic LBBC. Both paired cfDNA and primary/metastatic breast tumor samples showed hypermethylation of WNT1. TCGA analysis revealed significant WNT1 hypermethylation in the primary tumors of luminal breast cancer patients, with a negative association between WNT1 methylation and gene expression. In this proof-of-principle study, we discovered an episignature associated with metastatic LBBC using a genome-wide cfDNA methylation approach. We also identified the promoter hypermethylation of WNT1 in cfDNA as a potential noninvasive biomarker for luminal breast cancer. Our results support the use of EPIC arrays to identify new epigenetic noninvasive biomarkers in breast cancer.

Liquid Biopsy: A New Tool for Overcoming CDKi Resistance Mechanisms in Luminal Metastatic Breast Cancer.

Breast cancer (BC) is the most common cancer diagnosed in women worldwide. Approximately 70% of BC patients have the luminal subtype, which expresses hormone receptors (HR+). Adjuvant endocrine treatments are the standard of care for HR+/HER2- BC patients. Over time, approximately 30% of those patients develop endocrine resistance and metastatic disease. Cyclin-dependent kinase inhibitors (CDKi), in combination with an aromatase inhibitor or fulvestrant, have demonstrated superior efficacies in increasing progression-free survival, with a safe toxicity profile, in HR+/HER2- metastatic BC patients. CDKi blocks kinases 4/6, preventing G1/S cell cycle transition. However, not all of the patients respond to CDKi, and those who do respond ultimately develop resistance to the combined therapy. Studies in tumour tissues and cell lines have tried to elucidate the mechanisms that underlie this progression, but there are still no conclusive data. Over the last few years, liquid biopsy has contributed relevant information. Circulating tumour materials are potential prognostic markers for determining patient prognosis in metastatic luminal BC, for monitoring disease, and for treatment selection. This review outlines the different studies performed using liquid biopsy in patients with HR+ metastatic BC treated with CDKi plus endocrine therapy. We mainly focus on those studies that describe the possible resistance mechanisms in circulating tumour-derived material.

Short-Term Ex Vivo Culture of CTCs from Advance Breast Cancer Patients: Clinical Implications.

BACKGROUND: Circulating tumor cells (CTC) have relevance as prognostic markers in breast cancer. However, the functional properties of CTCs or their molecular characterization have not been well-studied. Experimental models indicate that only a few cells can survive in the circulation and eventually metastasize. Thus, it is essential to identify these surviving cells capable of forming such metastases. METHODS: We isolated viable CTCs from 50 peripheral blood samples obtained from 35 patients with advanced metastatic breast cancer using RosetteSepTM for ex vivo culture. The CTCs were seeded and monitored on plates under low adherence conditions and with media supplemented with growth factors and Nanoemulsions. Phenotypic analysis was performed by immunofluorescence and gene expression analysis using RT-PCR and CTCs counting by the Cellsearch® system. RESULTS: We found that in 75% of samples the CTC cultures lasted more than 23 days, predicting a shorter Progression-Free Survival in these patients, independently of having ≥5 CTC by Cellsearch®. We also observed that CTCs before and after culture showed a different gene expression profile. CONCLUSIONS: the cultivability of CTCs is a predictive factor. Furthermore, the subset of cells capable of growing ex vivo show stem or mesenchymal features and may represent the CTC population with metastatic potential in vivo.

Longitudinal CTCs gene expression analysis on metastatic castration-resistant prostate cancer patients treated with docetaxel reveals new potential prognosis markers.

CTCs have extensively been used for the monitoring and characterization of metastatic prostate cancer, but their application in the clinic is still very scarce. Besides, the resistance mechanisms linked to prostate cancer treatment remain unclear. Liquid biopsies represent the most promising alternative due to the complexity of biopsying bone metastasis and the duration of the disease. We performed a prospective longitudinal study in CTCs from 20 castration-resistant prostate cancer patients treated with docetaxel. For that, we used CellSearch® technology and a custom gene expression panel with qRT-PCR using a CTCs negative enrichment approach. We found that CTCs showed a hybrid phenotype during the disease, where epithelial features were associated with the presence of ≥ 5 CTCs/7.5 mL of blood, while high relative expression of the gene MYCL was observed preferentially in the set of samples with < 5 CTCs/7.5 mL of blood. At baseline, patients whose CTCs had stem or hybrid features showed a later progression. After 1 cycle of docetaxel, high relative expression of ZEB1 indicated worse outcome, while KRT19 and KLK3 high expression could predisposed the patients to a worse prognosis at clinical progression. In the present work we describe biomarkers with clinical relevance for the prediction of early response or resistance in castration-resistant prostate cancer patients. Besides, we question the utility of targeted isolated CTCs and the use of a limited number of markers to define the CTCs population.

Gene expression profiling of mouse p53-deficient epidermal carcinoma defines molecular determinants of human cancer malignancy.

BACKGROUND: The epidermal specific ablation of Trp53 gene leads to the spontaneous development of aggressive tumors in mice through a process that is accelerated by the simultaneous ablation of Rb gene. Since alterations of p53-dependent pathway are common hallmarks of aggressive, poor prognostic human cancers, these mouse models can recapitulate the molecular features of some of these human malignancies. RESULTS: To evaluate this possibility, gene expression microarray analysis was performed in mouse samples. The mouse tumors display increased expression of cell cycle and chromosomal instability associated genes. Remarkably, they are also enriched in human embryonic stem cell gene signatures, a characteristic feature of human aggressive tumors. Using cross-species comparison and meta-analytical approaches, we also observed that spontaneous mouse tumors display robust similarities with gene expression profiles of human tumors bearing mutated TP53, or displaying poor prognostic outcome, from multiple body tissues. We have obtained a 20-gene signature whose genes are overexpressed in mouse tumors and can identify human tumors with poor outcome from breast cancer, astrocytoma and multiple myeloma. This signature was consistently overexpressed in additional mouse tumors using microarray analysis. Two of the genes of this signature, AURKA and UBE2C, were validated in human breast and cervical cancer as potential biomarkers of malignancy. CONCLUSIONS: Our analyses demonstrate that these mouse models are promising preclinical tools aimed to search for malignancy biomarkers and to test targeted therapies of prospective use in human aggressive tumors and/or with p53 mutation or inactivation.

Analysis of a Real-World Cohort of Metastatic Breast Cancer Patients Shows Circulating Tumor Cell Clusters (CTC-clusters) as Predictors of Patient Outcomes.

Circulating tumor cell (CTC) enumeration has emerged as a powerful biomarker for the assessment of prognosis and the response to treatment in metastatic breast cancer (MBC). Moreover, clinical evidences show that CTC-cluster counts add prognostic information to CTC enumeration, however, their significance is not well understood, and more clinical evidences are needed. We aim to evaluate the prognostic value of longitudinally collected single CTCs and CTC-clusters in a heterogeneous real-world cohort of 54 MBC patients. Blood samples were longitudinally collected at baseline and follow up. CTC and CTC-cluster enumeration was performed using the CellSearch® system. Associations with progression-free survival (PFS) and overall survival (OS) were evaluated using Cox proportional hazards modelling. Elevated CTC counts and CTC-clusters at baseline were significantly associated with a shorter survival time. In joint analysis, patients with high CTC counts and CTC-cluster at baseline were at a higher risk of progression and death, and longitudinal analysis showed that patients with CTC-clusters had significantly shorter survival compared to patients without clusters. Moreover, patients with CTC-cluster of a larger size were at a higher risk of death. A longitudinal analysis of a real-world cohort of MBC patients indicates that CTC-clusters analysis provides additional prognostic value to single CTC enumeration, and that CTC-cluster size correlates with patient outcome.

Looking for a Better Characterization of Triple-Negative Breast Cancer by Means of Circulating Tumor Cells.

Traditionally, studies to address the characterization of mechanisms promoting tumor aggressiveness and progression have been focused only on primary tumor analyses, which could provide relevant information but have limitations to really characterize the more aggressive tumor population. To overcome these limitations, circulating tumor cells (CTCs) represent a noninvasive and valuable tool for real-time profiling of disseminated tumor cells. Therefore, the aim of the present study was to explore the value of CTC enumeration and characterization to identify markers associated with the outcome and the aggressiveness of triple-negative breast cancer (TNBC). For that aim, the CTC population from 32 patients diagnosed with TNBC was isolated and characterized. This population showed important cell plasticity in terms of expression of epithelia/mesenchymal and stemness markers, suggesting the relevance of epithelial to mesenchymal transition (EMT) intermediate phenotypes for efficient tumor dissemination. Importantly, the CTC signature demonstrated prognostic value to predict the patients' outcome and pointed to a relevant role of tissue inhibitor of metalloproteinases 1 (TIMP1) and androgen receptor (AR) for TNBC biology. Furthermore, we also analyzed the usefulness of the AR and TIMP1 blockade to target TNBC proliferation and dissemination using in vitro and in vivo zebra fish and mouse models. Overall, the molecular characterization of CTCs from advanced TNBC patients identifies highly specific biomarkers with potential applicability as noninvasive prognostic markers and reinforced the value of TIMP1 and AR as potential therapeutic targets to tackle the most aggressive breast cancer.

Transgenic mice expressing constitutively active Akt in oral epithelium validate KLFA as a potential biomarker of head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a common human neoplasia, of poor prognosis and survival, which frequently displays Akt overactivation. Previously, we reported that mice expressing high levels of constitutively Akt activity (myrAkt) in oral epithelia develop lesions and tumors in the oral cavity. MATERIALS AND METHODS: Functional genomics of primary keratinocytes from different transgenic mouse lines and immunostaining of mouse and human samples were performed in order to identify and validate putative biomarkers of oral cancer progression. RESULTS: The expression of KLF4 was found to be increased only in tumor prone samples from mice bearing overactivation of Akt. Such increased expression was confirmed in oral dysplasias and tumors arising in those mice. Tissue microarray analysis of human samples confirmed the association between active Akt and increased KLF4 expression. CONCLUSION: These data support the notion that KLF4 is potentially a reliable marker of HNSCC, and that myrAkt transgenic mice are valuable tools for preclinical research of HNSCC.

CTCs Expression Profiling for Advanced Breast Cancer Monitoring.

The study of circulating tumor cells (CTCs) has a huge clinical interest in advance and metastatic breast cancer patients. However, many approaches are biased by the use of epithelial markers, which underestimate non-epithelial CTCs phenotypes. CTCs enumeration provides valuable prognostic information; however, molecular characterization could be the best option to monitor patients throughout the disease since it may provide more relevant clinical information to the physicians. In this work, we aimed at enumerating and performing a molecular characterization of CTCs from a cohort of 20 patients with metastatic breast cancer (MBC), monitoring the disease at different time points of the therapy, and at progression when it occurred. To this end, we used a CTC negative enrichment protocol that allowed us to recover a higher variety of CTCs phenotypes. With this strategy, we were able to obtain gene expression data from CTCs from all the patients. In addition, we found that high expression levels of PALB2 and MYC were associated with a worse outcome. Interestingly, we identified that CTCs with an EpCAMhighVIMlowALDH1A1high signature showed both shorter overall survival (OS) and progression-free survival (PFS), suggesting that CTCs with epithelial-stem features had the most aggressive phenotype.

Fast and efficient microfluidic cell filter for isolation of circulating tumor cells from unprocessed whole blood of colorectal cancer patients.

Liquid biopsy offers unique opportunities for low invasive diagnosis, real-time patient monitoring and treatment selection. The phenotypic and molecular profile of circulating tumor cells (CTCs) can provide key information about the biology of tumor cells, contributing to personalized therapy. CTC isolation is still challenging, mainly due to their heterogeneity and rarity. To overcome this limitation, a microfluidic chip for label-free isolation of CTCs from peripheral blood was developed. This device, the CROSS chip, captures CTCs based on their size and deformability with an efficiency of 70%. Using 2 chips, 7.5 ml of whole blood are processed in 47 minutes with high purity, as compared to similar technologies and assessed by in situ immunofluorescence. The CROSS chip performance was compared to the CellSearch system in a set of metastatic colorectal cancer patients, resulting in higher capture of DAPI+/CK+/CD45- CTCs in all individuals tested. Importantly, CTC enumeration by CROSS chip enabled stratification of patients with different prognosis. Lastly, cells isolated in the CROSS chip were lysed and further subjected to molecular characterization by droplet digital PCR, which revealed a mutation in the APC gene for most patient samples analyzed, confirming their colorectal origin and the versatility of the technology for downstream applications.

Opposing roles of PIK3CA gene alterations to EZH2 signaling in non-muscle invasive bladder cancer.

The high rates of tumor recurrence and progression represent a major clinical problem in non-muscle invasive bladder cancer. Previous data showed that EZH2-dependent signaling mediates these processes, whereas the frequent alterations of PIK3CA gene (copy gains and mutations) are predictive of reduced recurrence. Here we show, using clinical samples and bladder cancer cell lines, a functional interaction between EZH2- and PIK3CA-dependent signaling pathways. PIK3CA alterations mediated, on the one hand, the increased expression of two miRNAs, miR-101 and miR-138, which posttranscriptionally downregulate EZH2 expression. On the other hand, PIK3CA alterations facilitate the activation of Akt which phosphorylates EZH2 on Ser21, precluding the trimethylation of histone H3 in K27. Remarkably the increased expression of miR101 or miR138 and the expression of Ser21-phosphorylated EZH2 are good prognostic factors regarding non-muscle invasive bladder cancer recurrence and progression. Collectively, this study provides molecular evidences indicating that the gene expression rewiring occurring in primary bladder tumors, associated with increased EZH2 expression and activity and mediating the increased recurrence and progression risk, are prevented by PIK3CA-dependent signaling. This molecular process may have deep implications in the management of bladder cancer patients and in the design of novel molecularly targeted therapeutic approaches.

Deregulation of the pRb-E2F4 axis alters epidermal homeostasis and favors tumor development.

E2F/RB activity is altered in most human tumors. The retinoblastoma family of proteins plays a key role in regulating the progression of the cell cycle from the G1 to S phases. This is achieved through negative regulation of E2F transcription factors, important positive regulators of cell cycle entry. E2F family members are divided into two groups: activators (E2F1-E2F3a) and repressors (E2F3b-E2F8). E2F4 accounts for a large part of the E2F activity and is a main E2F repressor member in vivo. Perturbations in the balance from quiescence towards proliferation contribute to increased mitotic gene expression levels frequently observed in cancer. We have previously reported that combined Rb1-Rbl1 or Rb1-E2f1 ablation in epidermis produces important alterations in epidermal proliferation and differentiation, leading to tumor development. However, the possible roles of E2F4 in this context are still to be determined. Here, we show the absence of any discernible phenotype in the skin of mice lacking of E2f4. In contrast, the inducible loss of Rb1 in the epidermis of E2F4-null mice produced multiple skin abnormalities including altered differentiation and proliferation, spontaneous wounds, carcinoma in situ development and stem cell perturbations. All these phenotypic alterations are associated with extensive gene expression changes, the induction of c-myc and the Akt activation. Moreover the whole transcriptome analyses in comparison with previous models generated also revealed extensive changes in multiple repressive complexes and in transcription factor activity. These results point to E2F4 as a master regulator in multiple steps of epidermal homeostasis in Rb1 absence.