Diffuse coexpression of thyroid transcription factor-1 and p40 in a non-small cell carcinoma of the lung: Second case in the female population / Coexpresión difusa del factor de transcripción tiroidea 1 y p40 en un carcinoma de célula no pequeña del pulmón: segundo caso en la población de sexo femenino

Some non-small cell carcinomas of the lung can express TTF1 and p40 in the same tumor cells. This event has been described in only six cases prior to this one, and only in one other female. It is an extraordinary event that appears as a new entity yet to be defined. The case presented is a woman with a non-small cell lung carcinoma with diffuse coexpression of TTF1 and p40 in the same cells. (AU)

Algunos carcinomas de célula no pequeña del pulmón pueden expresar TTF1 y p40 en las mismas células tumorales. Este evento se ha descrito únicamente en 6 casos anteriores a este, y solo en otra persona del sexo femenino. Se trata de un evento extraordinario que se muestra como una nueva entidad todavía por definir. El caso que se presenta versa sobre una mujer con un carcinoma de pulmón de célula no pequeña con coexpresión difusa en las mismas células de TTF1 y p40. (AU)

Ex Vivo Culture of Circulating Tumor Cells in the Cerebral Spinal Fluid from Melanoma Patients to Study Melanoma-Associated Leptomeningeal Disease.

Melanoma-associated leptomeningeal disease (M-LMD) occurs when circulating tumor cells (CTCs) enter into the cerebral spinal fluid (CSF) and colonize the meninges, the membrane layers that protect the brain and the spinal cord. Once established, the prognosis for M-LMD patients is dismal, with overall survival ranging from weeks to months. This is primarily due to a paucity in our understanding of the disease and, as a consequence, the availability of effective treatment options. Defining the underlying biology of M-LMD will significantly improve the ability to adapt available therapies for M-LMD treatment or design novel inhibitors for this universally fatal disease. A major barrier, however, lies in obtaining sufficient quantities of CTCs from the patient-derived CSF (CSF-CTCs) to conduct preclinical experiments, such as molecular characterization, functional analysis, and in vivo efficacy studies. Culturing CSF-CTCs ex vivo has also proven to be challenging. To address this, a novel protocol for the culture of patient-derived M-LMD CSF-CTCs ex vivo and in vivo is developed. The incorporation of conditioned media produced by human meningeal cells (HMCs) is found to be critical to the procedure. Cytokine array analysis reveals that factors produced by HMCs, such as insulin-like growth factor-binding proteins (IGFBPs) and vascular endothelial growth factor-A (VEGF-A), are important in supporting CSF-CTC survival ex vivo. Here, the usefulness of the isolated patient-derived CSF-CTC lines is demonstrated in determining the efficacy of inhibitors that target the insulin-like growth factor (IGF) and mitogen-activated protein kinase (MAPK) signaling pathways. In addition, the ability to intrathecally inoculate these cells in vivo to establish murine models of M-LMD that can be employed for preclinical testing of approved or novel therapies is shown. These tools can help unravel the underlying biology driving CSF-CTC establishment in the meninges and identify novel therapies to reduce the morbidity and mortality associated with M-LMD.

Circulating Cancer-Associated Macrophage-like Cells as a Blood-Based Biomarker of Response to Immune Checkpoint Inhibitors.

Evidence has been provided that circulating cancer-associated macrophage-like cell (CAM-L) numbers increase in response to chemotherapy, with an inverse trend compared to circulating tumor cells (CTCs). In the era of evolving cancer immunotherapy, whether CAM-Ls might have a potential role as predictive biomarkers of response has been unexplored. We evaluated whether a serial blood evaluation of CTC to CAM-L ratio might predict response to immune checkpoint inhibitors in a cohort of non-small-cell lung cancer patients. At baseline, CTCs, CAM-Ls, and the CTC/CAM-L ratio significantly correlate with both progression-free survival (PFS) and overall survival (OS). The baseline CTC/CAM-L ratio was significantly different in early progressors (4.28 ± 3.21) compared to long responders (0.42 ± 0.47) (p = 0.001). In patients treated with immune checkpoint inhibitors, a CTC/CAM-L ratio ≤ 0.25 at baseline is associated with better PFS and OS. A baseline CTC/CAM-L ratio ≤ 0.25 is statistically significant to discriminate early progressions from durable response. The results of the present pilot study suggest that CAM-Ls together with CTCs could play an important role in evaluating patients treated with cancer immunotherapy.

ECL cytosensor for sensitive and label-free detection of circulating tumor cells based on hierarchical flower-like gold microstructures.

The development of sensitive and efficient cell sensing strategies to detect circulating tumor cells (CTCs) in peripheral blood is crucial for the early diagnosis and prognostic assessment of cancer clinical treatment. Herein, an array of hierarchical flower-like gold microstructures (HFGMs) with anisotropic nanotips was synthesized by a simple electrodeposition method and used as a capture substrate to construct an ECL cytosensor based on the specific recognition of target cells by aptamers. The complex topography of the HFGMs array not only catalyzed the enhancement of ECL signals, but also induced the cells to generate more filopodia, improving the capture efficiency and shortening the capture time. The effect of topographic roughness on cell growth and adhesion propensity was also investigated, while the cell capture efficiency was proposed to be an important indicator affecting the accuracy of the ECL cytosensor. In addition, the capture of cells on the electrode surface increased the steric hindrance, which caused ECL signal changes in the Ru(bpy)32+ and TPrA system, realizing the quantitative detection of MCF-7 cells. The detection range of the sensor was from 102 to 106 cells mL-1 and the detection limit was 18 cells mL-1. The proposed detection method avoids the process of separation, labeling and counting, which has great potential for sensitive detection in clinical applications.

Nanobiotechnology augmented cancer stem cell guided management of cancer: liquid-biopsy, imaging, and treatment.

Cancer stem cells (CSCs) represent both a key driving force and therapeutic target of tumoral carcinogenesis, tumor evolution, progression, and recurrence. CSC-guided tumor diagnosis, treatment, and surveillance are strategically significant in improving cancer patients' overall survival. Due to the heterogeneity and plasticity of CSCs, high sensitivity, specificity, and outstanding targeting are demanded for CSC detection and targeting. Nanobiotechnologies, including biosensors, nano-probes, contrast enhancers, and drug delivery systems, share identical features required. Implementing these techniques may facilitate the overall performance of CSC detection and targeting. In this review, we focus on some of the most recent advances in how nanobiotechnologies leverage the characteristics of CSC to optimize cancer diagnosis and treatment in liquid biopsy, clinical imaging, and CSC-guided nano-treatment. Specifically, how nanobiotechnologies leverage the attributes of CSC to maximize the detection of circulating tumor DNA, circulating tumor cells, and exosomes, to improve positron emission computed tomography and magnetic resonance imaging, and to enhance the therapeutic effects of cytotoxic therapy, photodynamic therapy, immunotherapy therapy, and radioimmunotherapy are reviewed.

Liquid biopsy techniques and lung cancer: diagnosis, monitoring and evaluation.

Lung cancer stands as the most prevalent form of cancer globally, posing a significant threat to human well-being. Due to the lack of effective and accurate early diagnostic methods, many patients are diagnosed with advanced lung cancer. Although surgical resection is still a potential means of eradicating lung cancer, patients with advanced lung cancer usually miss the best chance for surgical treatment, and even after surgical resection patients may still experience tumor recurrence. Additionally, chemotherapy, the mainstay of treatment for patients with advanced lung cancer, has the potential to be chemo-resistant, resulting in poor clinical outcomes. The emergence of liquid biopsies has garnered considerable attention owing to their noninvasive nature and the ability for continuous sampling. Technological advancements have propelled circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), extracellular vesicles (EVs), tumor metabolites, tumor-educated platelets (TEPs), and tumor-associated antigens (TAA) to the forefront as key liquid biopsy biomarkers, demonstrating intriguing and encouraging results for early diagnosis and prognostic evaluation of lung cancer. This review provides an overview of molecular biomarkers and assays utilized in liquid biopsies for lung cancer, encompassing CTCs, ctDNA, non-coding RNA (ncRNA), EVs, tumor metabolites, TAAs and TEPs. Furthermore, we expound on the practical applications of liquid biopsies, including early diagnosis, treatment response monitoring, prognostic evaluation, and recurrence monitoring in the context of lung cancer.

Circulating tumor cell-derived exosome-transmitted long non-coding RNA TTN-AS1 can promote the proliferation and migration of cholangiocarcinoma cells.

BACKGROUND: Exosomes assume a pivotal role as essential mediators of intercellular communication within tumor microenvironments. Within this context, long noncoding RNAs (LncRNAs) have been observed to be preferentially sorted into exosomes, thus exerting regulatory control over the initiation and progression of cancer through diverse mechanisms. RESULTS: Exosomes were successfully isolated from cholangiocarcinoma (CCA) CTCs organoid and healthy human serum. Notably, the LncRNA titin-antisense RNA1 (TTN-AS1) exhibited a conspicuous up-regulation within CCA CTCs organoid derived exosomes. Furthermore, a significant elevation of TTN-AS1 expression was observed in tumor tissues, as well as in blood and serum exosomes from patients afflicted with CCA. Importantly, this hightened TTN-AS1 expression in serum exosomes of CCA patients manifested a strong correlation with both lymph node metastasis and TNM staging. Remarkably, both CCA CTCs organoid-derived exosomes and CCA cells-derived exosomes featuring pronounced TTN-AS1 expression demonstrated the capability to the proliferation and migratory potential of CCA cells. Validation of these outcomes was conducted in vivo experiments. CONCLUSIONS: In conclusion, our study elucidating that CCA CTCs-derived exosomes possess the capacity to bolster the metastasis tendencies of CCA cells by transporting TTN-AS1. These observations underscore the potential of TTN-AS1 within CTCs-derived exosomes to serve as a promising biomarker for the diagnosis and therapeutic management of CCA.

Detecting androgen receptor (AR), AR variant 7 (AR-V7), prostate-specific membrane antigen (PSMA), and prostate-specific antigen (PSA) gene expression in CTCs and plasma exosome-derived cfRNA in patients with metastatic castration-resistant prostate cancer (mCRPC) by integrating the VTX-1 CTC isolation system with the QIAGEN AdnaTest.

BACKGROUND: Therapies for metastatic castration-resistant prostate cancer (mCRPC) include targeting the androgen receptor (AR) with androgen receptor inhibitors (ARIs) and prostate-specific membrane antigen (PSMA). Having the ability to detect AR, AR splice variant 7 (AR-V7), or PSMA in circulating tumor cells (CTCs) or circulating exosomal cell-free RNA (cfRNA) could be helpful to guide selection of the appropriate therapy for each individual patient. The Vortex Biosciences VTX-1 system is a label-free CTC isolation system that enables the detection of the expression of multiple genes in both CTCs and exosomal cfRNA from the same blood sample in patients with mCRPC. Detection of both AR-V7 and PSMA gene expression in both CTCs and cfRNA simultaneously has not yet been reported. METHODS: To characterize the combined VTX-1-AdnaDetect workflow, 22Rv1 cancer cells were spiked into blood from healthy donors and processed with the VTX-1 to mimic patient samples and assess performances (capture efficiency, purity, AR and AR-V7 expression). Then, we collected 19 blood samples from 16 patients with mCRPC and therapeutic resistance to androgen receptor inhibitors (ARIs). Plasma was separated and the plasma-depleted blood was processed further with the VTX-1 to collect CTCs. Both plasma exosomal cfRNA and CTCs were subsequently analyzed for AR, AR-V7, PSMA, and prostate-specific antigen (PSA) mRNA expression using the AdnaTest ProstateCancerPanel AR-V7 assay. RESULTS: AR-V7 expression could be detected in 22Rv1 cells spiked into blood from healthy volunteers as well as in CTCs and plasma-derived exosomal cfRNA from patients with mCRPC by processing blood with the VTX-1 CTC isolation system followed by the AdnaTest ProstateCancerPanel AR-V7 assay. 94.7% of patient blood samples (18/19) had detectable AR expression in either CTCs or exosomal cfRNA (16 in CTCs, 12 in cfRNA). 15.8% of the 19 patient blood samples (3/19) were found to have AR-V7-positive (AR-V7+) CTCs, one of which was also AR-V7+ in the exosomal cfRNA analysis. 42.1% of patient blood samples (8/19) were found to be PSMA positive (PSMA+): 26.3% (5/19) were PSMA+ in the CTC analysis and 31.6% (6/19) were PSMA+ in the exosomal cfRNA analysis. Of those 8 PSMA+ samples, 2 had detectable PSMA only in CTCs, and 3 had detectable PSMA only in exosomal cfRNA. CONCLUSION: VTX-1 enables isolation of CTCs and plasma exosomes from a single blood draw and can be used for detecting AR-V7 and PSMA mRNA in both CTCs and cfRNA in patients with mCRPC and resistance to ARIs. This technology facilitates combining RNA measurements in CTCs and exosomal cfRNA for future studies to develop potentially clinically relevant cancer biomarker detection in blood.

Circulating tumor cells shielded with extracellular vesicle-derived CD45 evade T cell attack to enable metastasis.

Circulating tumor cells (CTCs) are precursors of distant metastasis in a subset of cancer patients. A better understanding of CTCs heterogeneity and how these CTCs survive during hematogenous dissemination could lay the foundation for therapeutic prevention of cancer metastasis. It remains elusive how CTCs evade immune surveillance and elimination by immune cells. In this study, we unequivocally identified a subpopulation of CTCs shielded with extracellular vesicle (EVs)-derived CD45 (termed as CD45+ CTCs) that resisted T cell attack. A higher percentage of CD45+ CTCs was found to be closely correlated with higher incidence of metastasis and worse prognosis in cancer patients. Moreover, CD45+ tumor cells orchestrated an immunosuppressive milieu and CD45+ CTCs exhibited remarkably stronger metastatic potential than CD45- CTCs in vivo. Mechanistically, CD45 expressing on tumor surfaces was shown to form intercellular CD45-CD45 homophilic interactions with CD45 on T cells, thereby preventing CD45 exclusion from TCR-pMHC synapse and leading to diminished TCR signaling transduction and suppressed immune response. Together, these results pointed to an underappreciated capability of EVs-derived CD45-dressed CTCs in immune evasion and metastasis, providing a rationale for targeting EVs-derived CD45 internalization by CTCs to prevent cancer metastasis.

Orthotopic model for the analysis of melanoma circulating tumor cells.

Metastatic melanoma, a highly lethal form of skin cancer, presents significant clinical challenges due to limited therapeutic options and high metastatic capacity. Recent studies have demonstrated that cancer dissemination can occur earlier, before the diagnosis of the primary tumor. The progress in understanding the kinetics of cancer dissemination is limited by the lack of animal models that accurately mimic disease progression. We have established a xenograft model of human melanoma that spontaneously metastasizes to lymph nodes and lungs. This model allows precise monitoring of melanoma progression and is suitable for the quantitative and qualitative analysis of circulating tumor cells (CTCs). We have validated a flow cytometry-based protocol for CTCs enumeration and isolation. We could demonstrate that (i) CTCs were detectable in the bloodstream from the fourth week after tumor initiation, coinciding with the lymph node metastases appearance, (ii) excision of the primary tumor accelerated the formation of metastases in lymph nodes and lungs as early as one-week post-surgery, accompanied by the increased numbers of CTCs, and (iii) CTCs change their surface protein signature. In summary, we present a model of human melanoma that can be effectively utilized for future drug efficacy studies.

Effect of acute inflammatory reaction induced by biopsy on tumor microenvironment.

When it comes to the diagnosis of solid tumors, biopsy is always the gold standard. However, traumatic and inflammatory stimuli are so closely related to tumor initiation and development that the acute inflammatory response induced by biopsy can give rise to changes in the tumor microenvironment, including recruitment of immunosuppressive cells (M2 macrophages, Treg cells, Tumor-associated neutrophils) and secretion of inflammation-associated cytokines, to create immunosuppressive conditions that enable the increase of circulating tumor cells in the peripheral circulation and promote the metastatic spread of tumors after surgery. In this review, we discuss dynamic changes and inhibitory characteristics of biopsy on tumor microenvironment. By investigating its mechanism of action and summarizing the current therapeutic strategies for biopsy-induced tumor immunosuppressive microenvironment, the future of using biopsy-induced inflammation to improve the therapeutic effects and prognosis of patients is prospected.

A novel method for the isolation of single cells mimicking circulating tumour cells adhered on Smart Bio Surface slides by Laser Capture Microdissection.

In recent years, the importance of isolating single cells from blood circulation for several applications, such as non-invasive tumour diagnosis, the monitoring of minimal residual disease, and the analysis of circulating fetal cells for prenatal diagnosis, urged the need to set up innovative methods. For such applications, different methods were developed. All show some weaknesses, especially a limited sensitivity, and specificity. Here we present a new method for isolating a single or a limited number of cells adhered to SBS slides (Tethis S.p.a.) (a glass slide coated with Nanostructured Titanium Dioxide) by Laser Capture Microdissection (LCM) and subsequent Whole Genome Amplification. SBS slides have been shown to have an optimal performance in immobilizing circulating tumour cells (CTCs) from early breast cancer patients. In this work, we spiked cancer cells in blood samples to mimic CTCs. By defining laser parameters to cut intact samples, we were able to isolate genetically intact single cells. We demonstrate that SBS slides are optimally suited for isolating cells using LCM and that this method provides high-quality DNA, ideal for gene-specific assays such as PCR and Sanger sequencing for mutation analysis.

CTC-Race: Single-Cell Motility Assay of Circulating Tumor Cells from Metastatic Lung Cancer Patients.

Distinctive subpopulations of circulating tumor cells (CTCs) with increased motility are considered to possess enhanced tumor-initiating potential and contribute to metastasis. Single-cell analysis of the migratory CTCs may increase our understanding of the metastatic process, yet most studies are limited by technical challenges associated with the isolation and characterization of these cells due to their extreme scarcity and heterogeneity. We report a microfluidic method based on CTCs' chemotactic motility, termed as CTC-Race assay, that can analyze migrating CTCs from metastatic non-small-cell lung cancer (NSCLC) patients with advanced tumor stages and enable concurrent biophysical and biochemical characterization of them with single-cell resolution. Analyses of motile CTCs in the CTC-Race assay, in synergy with other single cell characterization techniques, could provide insights into cancer metastasis.

Deep learning-enabled detection of rare circulating tumor cell clusters in whole blood using label-free, flow cytometry.

Metastatic tumors have poor prognoses for progression-free and overall survival for all cancer patients. Rare circulating tumor cells (CTCs) and rarer circulating tumor cell clusters (CTCCs) are potential biomarkers of metastatic growth, with CTCCs representing an increased risk factor for metastasis. Current detection platforms are optimized for ex vivo detection of CTCs only. Microfluidic chips and size exclusion methods have been proposed for CTCC detection; however, they lack in vivo utility and real-time monitoring capability. Confocal backscatter and fluorescence flow cytometry (BSFC) has been used for label-free detection of CTCCs in whole blood based on machine learning (ML) enabled peak classification. Here, we expand to a deep-learning (DL)-based, peak detection and classification model to detect CTCCs in whole blood data. We demonstrate that DL-based BSFC has a low false alarm rate of 0.78 events per min with a high Pearson correlation coefficient of 0.943 between detected events and expected events. DL-based BSFC of whole blood maintains a detection purity of 72% and a sensitivity of 35.3% for both homotypic and heterotypic CTCCs starting at a minimum size of two cells. We also demonstrate through artificial spiking studies that DL-based BSFC is sensitive to changes in the number of CTCCs present in the samples and does not add variability in detection beyond the expected variability from Poisson statistics. The performance established by DL-based BSFC motivates its use for in vivo detection of CTCCs. Using transfer learning, we additionally validate DL-based BSFC on blood samples from different species and cancer cell types. Further developments of label-free BSFC to enhance throughput could lead to critical applications in the clinical detection of CTCCs and ex vivo isolation of CTCC from whole blood with minimal disruption and processing steps.

Investigating the role of circulating tumor cells in gastric cancer: a comprehensive systematic review and meta-analysis.

Investigating the role of circulating tumor cells (CTCs) and their characteristics is still controversial in patients with gastric cancer (GC). Therefore, in this study, to provide a comprehensive review and meta-analyses of the literature on association of CTCs with gastric cancer, Scopus, Web of Science, Embase, and Medline were searched for systematic reviews and meta-analyses conducted during February 2022 using the keywords. Risk of bias, hazard ratios (HRs), and risk differences (RD) were assessed. Forty-five studies containing 3,342 GC patients from nine countries were assessed. The overall prevalence of CTC in GC was 69.37% (60.27, 77.78). The pooled result showed that increased mortality in GC patients was significantly associated with positive CTCs, poor overall survival (HR = 2.73, 95%CI 2.34-3.24, p < 0.001), and progression-free survival rate (HR = 2.78, 95%CI 2.01-3.85, p < 0.001). Subgroup analyses regarding markers, detection methods, treatment type, presence of distance metastasis, presence of lymph node metastasis, and overall risk of bias showed significant associations between the groups in terms of the incidence rates of CTCs, OS, and PFS. In addition, the results of risk differences based on sampling time showed that the use of the cell search method (RD: - 0.19, 95%CI (- 0.28, - 0.10), p < 0.001), epithelial marker (RD: - 0.12, 95%CI (- 0.25, 0.00), p 0.05) and mesenchymal markers (RD: - 0.35, 95%CI (- 0.57, - 0.13), p 0.002) before the treatment might have a higher diagnostic power to identify CTCs and also chemotherapy treatment (RD: - 0.17, 95%CI (- 0.31, - 0.03), p 0.016) could significantly reduce the number of CTCs after the treatment. We also found that the risk differences between the clinical early and advanced stages were not statistically significant (RD: - 0.10, 95%CI (- 0.23, 0.02), P 0.105). Also, in the Lauren classification, the incidence of CTC in the diffuse type (RD: - 0.19, 95%CI (- 0.37, - 0.01), P0.045) was higher than that in the intestinal type. Meta-regression analysis showed that baseline characteristics were not associated with the detection of CTCs in GC patients. According to our systematic review and meta-analysis, CTCs identification may be suggested as a diagnostic technique for gastric cancer screening, and the outcomes of CTC detection may also be utilized in the future to create personalized medicine programs.

Isolating Circulating Cancer Stem Cells (CCSCs) from Human Whole Blood.

Measuring circulating tumor cells (CTCs) or circulating cancer stem cells (CCSCs) in blood, which shed from primary tumors, is a noninvasive method to screen and/or diagnose patients with a high risk of developing metastatic cancers or relapse. Here, we describe an optimized and standardized laboratory method for isolating CCSCs from human blood samples, using cancer-specific stem cell biomarkers (Kantara et al., Lab Invest 95:100-112, 2015). When performing this technique, 0-1 circulating epithelial tumor cells/mL blood should be expected in patients free of malignant adenocarcinomas compared to over 3 circulating cancer stem cells/mL blood in patients positive for malignant adenocarcinomas (Kantara et al., Lab Invest 95:100-112, 2015).

Liquid Biopsy: A Game Changer for Type 2 Diabetes.

As the burden of type 2 diabetes (T2D) continues to escalate globally, there is a growing need for novel, less-invasive biomarkers capable of early diabetes detection and monitoring of disease progression. Liquid biopsy, recognized for its minimally invasive nature, is increasingly being applied beyond oncology, and nevertheless shows its potential when the collection of the tissue biopsy is not possible. This diagnostic approach involves utilizing liquid biopsy markers such as cell-free nucleic acids, extracellular vesicles, and diverse metabolites for the molecular diagnosis of T2D and its related complications. In this context, we thoroughly examine recent developments in T2D liquid biopsy research. Additionally, we discuss the primary challenges and future prospects of employing liquid biopsy in the management of T2D. Prognosis, diagnosis and monitoring of T2D through liquid biopsy could be a game-changing technique for personalized diabetes management.