Flow cytometry potential applications in characterizing solid tumors main phenotype, heterogeneity and circulating cells

Flow cytometry (FCM) is a unique technique that allows rapid quantitative measurement of multiple parameters on a large number of cells at the individual level. FCM is based on immunolabelling with fluorochrome-conjugated antibodies, leading to high sensitivity and precision while time effective sample preparation. FCM can be performed on tissue following enzymatic or mechanical dissociation. The expression of epithelial antigens and cytokeratin isoforms help in distinguishing tumor cells from adjacent epithelial cells and from tumor infiltrating leukocytes. Tumor phenotypes can be characterized on expression intensity, aberrancies and presence of tumor-associated antigens as well as their cell proliferation rate and eventual heteroploidy. FCM can measure quantitative expression of hormone or growth factor receptors, immunoregulatory proteins to guide adjuvant therapy. Expression of adhesion molecules tells on tumor’s capacity for tissue invasion and metastasis seeding. Tumor heterogeneity can be explored quantitatively and rare, potentially emerging, clones with poor prognosis can be detected. FCM is easily applicable on fine needle aspiration and in any tumor related biological fluids. FCM can also be used to detect circulating tumor cells (CTC) to assess metastatic potential at diagnosis or during treatment. Detecting CTC could allow early detection of tumors before they are clinically expressed although some difficulties still need to be solved. It thus appears that FCM should be in the pathologist tool box to improve cancer diagnosis, classification and prognosis evaluation as well as in orientating personalized adjuvant therapy and immunotherapy. More developments are still required to better known tumor phenotypes and their potential invasiveness.

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Flow Cytometry of Nonhematopoietic Neoplasms

Acta Cytologica ◽

10.1159/000448371 ◽

2016 ◽

Vol 60 (4) ◽

pp. 336-343 ◽

Cited By ~ 14

Author(s):

Vinodh Pillai ◽

David M. Dorfman

Keyword(s):

Flow Cytometry ◽

Wilms Tumor ◽

Flow Cytometric Analysis ◽

Germ Cell Tumors ◽

High Sensitivity ◽

Needle Aspiration ◽

Color Flow ◽

Flow Cytometric ◽

Epithelial Antigens ◽

Epithelial Neoplasms

Many epithelial neoplasms can be analyzed by flow cytometry (FC), particularly from serous cavity effusion samples, using EpCAM, a cell adhesion molecule expressed on most normal epithelial cells and expressed at a higher level in most epithelial neoplasms. A simple 3-color flow cytometric panel can provide a high sensitivity and specificity compared to cytomorphology. FC provides more rapid immunophenotyping than conventional immunohistochemical staining, can identify rare malignant cells that could be missed by a cytological exam alone, and can be utilized to evaluate limited samples such as cerebrospinal fluid or fine-needle aspiration samples. Flow cytometric analysis for epithelial antigens can be combined with DNA ploidy analysis or assessment of the nucleus-to-cytoplasm ratio. Panels of flow cytometric markers are useful for the assessment of pediatric nonhematopoietic neoplasms, including neuroblastomas, primitive neuroectodermal tumors, Wilms' tumor, rhabdomyosarcomas, germ cell tumors, and hemangiopericytomas, as well as small-round-blue-cell tumors in adults, including small-cell carcinomas.

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In Vivo Lymphatic Circulating Tumor Cells and Progression of Metastatic Disease

Cancers ◽

10.3390/cancers12102866 ◽

2020 ◽

Vol 12 (10) ◽

pp. 2866

Author(s):

Mikyung Han ◽

Julia Alex Watts ◽

Azemat Jamshidi-Parsian ◽

Urooba Nadeem ◽

Mustafa Sarimollaoglu ◽

...

Keyword(s):

Flow Cytometry ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Metastatic Disease ◽

Primary Tumor ◽

High Sensitivity ◽

Disease Stage ◽

Primary Tumors ◽

Integrative Assessment

The dissemination of circulating tumor cells (CTCs) by lymph fluid is one of the key events in the development of tumor metastasis. However, little progress has been made in studying lymphatic CTCs (L-CTCs). Here, we demonstrate the detection of L-CTCs in preclinical mouse models of melanoma and breast cancer using in vivo high-sensitivity photoacoustic and fluorescent flow cytometry. We discovered that L-CTCs are be detected in pre-metastatic disease stage. The smallest primary tumor that shed L-CTCs was measured as 0.094mm×0.094mm, its volume was calculated as 0.0004 mm3; and its productivity was estimated as 1 L-CTC per 30 minutes. As the disease progressed, primary tumors continued releasing L-CTCs with certain individual dynamics. The integrated assessment of lymph and blood underlined the parallel dissemination of CTCs at all disease stages. However, the analysis of links between L-CTC counts, blood CTC (B-CTC) counts, primary tumor size and metastasis did not reveal statistically significant correlations, likely due to L-CTC heterogeneity. Altogether, our results showed the feasibility of our diagnostic platform using photoacoustic flow cytometry for preclinical L-CTC research with translational potential. Our findings also demonstrated new insights into lymphatic system involvement in CTC dissemination. They help to lay the scientific foundation for the consideration of L-CTCs as prognostic markers of metastasis and to emphasize the integrative assessment of lymph and blood.

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Clinical Utility of Endoscopic Ultrasound-Guided Fine-Needle Aspiration Biopsy In Mixed Ductal-Neuroendocrine Neoplasms of The Pancreas

10.21203/rs.3.rs-207925/v1 ◽

2021 ◽

Author(s):

pengyan Wang ◽

Yan Wu ◽

Jingci Chen ◽

Ying Jiang ◽

Xiaowei Xue ◽

...

Keyword(s):

Adjuvant Therapy ◽

Endoscopic Ultrasound ◽

Fine Needle Aspiration ◽

High Sensitivity ◽

Needle Aspiration ◽

Ductal Adenocarcinoma ◽

Neuroendocrine Neoplasms ◽

Ultrasound Guided ◽

Fine Needle ◽

Pancreatic Masses

Abstract Background: Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) biopsy is the first line diagnostic tool used in the evaluation of solid pancreatic masses with high sensitivity and specificity prior to the surgery. Mixed ductal-neuroendocrine neoplasms of the pancreas are rare entity consisting of both ductal adenocarcinoma and neuroendocrine neoplasms, with each component representing at least 30%. Methods: We performed a retrospective analysis of patients with a diagnosis of pancreatic mixed ductal adenocarcinoma and neuroendocrine neoplasms by EUS-FNA specimens at Peking Union Medical College Hospital between 2010-2019 and presented a review of the literature.Results: A total of 3 cases (1 male and 2 females) were evaluated, aged 46, 38 and 62 years, and presented with 3-cm, 2.3-cm and 1.8-cm pancreatic masses, respectively. All the patients presented with abdominal pain and underwent EUS-FNA biopsy. FNA smears revealed a mixture of ductal adenocarcinoma and neuroendocrine neoplasms, identified by immunohistochemistry. According to Ki-67 proliferation index and mitotic count, the tumor grades of the neuroendocrine component were classified as NET G2 in 2 cases (cases 2 and 3) and NEC in 1 (case 1). All of the patients received adjuvant therapy. The diagnosis of patient 1 was confirmed on surgical excision, and she died 5 months after surgery due to hepatic metastases. The other two patients did not undergo surgery, but achieved long – term survival of 3 and 5 years, respectively. To date, 34 cases of pancreatic mixed ductal adenocarcinoma and neuroendocrine neoplasms have been reported in the English language literature including our present three cases. Among them, 5 of 12 patients who underwent preoperative biopsy were diagnosed as combined ductal adenocarcinoma and neuroendocrine neoplasms, of which 3 surgical resection specimens available were identified as mixed ductal-neuroendocrine neoplasms.Conclusion: We suggest that EUS-FNA biopsy is the most effective method for rapid evaluation of pancreatic mixed ductal-neuroendocrine neoplasms with high sensitivity, which is important to determine the most optimum therapy. If the tumor grades of the neuroendocrine component were classified as NET G2 by EUS-FNA biopsy, we propose appropriate adjuvant therapy rather than surgery.

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Sequential detection of mRNA for the chemokine IL-8 and macrophages (F4/80) in human melanoma

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010014141x ◽

1995 ◽

Vol 53 ◽

pp. 1008-1009

Author(s):

C.D. Bucana ◽

R. Sanchez ◽

R. Singh ◽

I.J. Fidler

Keyword(s):

Tumor Cells ◽

Nude Mice ◽

Constitutive Expression ◽

Metastatic Potential ◽

Melanoma Cells ◽

Human Melanoma ◽

Angiogenic Factor ◽

Infiltrating Cells ◽

Immunoperoxidase Assay ◽

Multifunctional Cytokine

The purpose of this study was to demonstrate by ISH the presence of IL-8 mRNA, and by immunohistochemistry (IHC) the presence of the chemokine IL-8 and the distribution of infiltrating macrophages in subcutaneous melanomas in the same tumor. IL-8 is a multifunctional cytokine produced by melanoma cells, activated macrophages and monocytes and it has been shown to be a growth and angiogenic factor for tumor cells. More recently it was shown that constitutive expression of IL-8 correlated directly with metastatic potential of human melanoma cells in nude mice. IL-8 content of a solid tumor as determined by Western blot analysis does not take into account the contribution of macrophages. Previous studies showed that murine tumors contain many infiltrating cells interspersed among tumor cells whereas human tumors growing in nude mice exhibit macrophages at the periphery or between tumor islands. In this study we demonstrate the expression of IL-8 and the distribution of macrophages by immunoperoxidase assay and IL-8 mRNA by ISH.

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Role of exosomes in diagnosis and therapy of prostate cancer

I P Pavlov Russian Medical Biological Herald ◽

10.23888/pavlovj2020283399-405 ◽

2020 ◽

Vol 28 (3) ◽

pp. 399-405

Author(s):

Fabrizio Fontana ◽

Olga A. Babenko

Keyword(s):

Prostate Cancer ◽

Cancer Cells ◽

Tumor Cells ◽

Biological Fluids ◽

Diagnosis And Treatment ◽

Diagnosis And Therapy ◽

The Future ◽

Important Direction ◽

Potential Biomarkers

Aim of this letter is to attract the attention of journal readers to the study of exosomes as an important direction in the development of Oncology, in particular, in the diagnosis and treatment of prostate cancer. Exosomes are produced by tumor cells and regulate proliferation, metastasis, and the development of chemoresistance. Their extraction from biological fluids allows further use of these vesicles as potential biomarkers of prostate cancer. In the future, exosomes can be successfully used in the delivery of drugs and other anti-tumor substances to cancer cells.

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Evaluation of Production Lots of a Rapid Point-of-Care Lateral Flow Serological Test Intended for Identification of IgM and IgG against the N-Terminal Part of the Spike Protein (S1) of SARS-CoV-2

Viruses ◽

10.3390/v13061043 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1043

Author(s):

Tove Hoffman ◽

Linda Kolstad ◽

Bengt Rönnberg ◽

Åke Lundkvist

Keyword(s):

Predictive Value ◽

Serological Test ◽

Point Of Care ◽

External Validation ◽

High Sensitivity ◽

Lateral Flow ◽

Spike Protein ◽

Individual Level ◽

Lateral Flow Test ◽

Production Errors

The potential of rapid point-of-care (POC) tests has been subject of doubt due to an eventual risk of production errors. The aim was therefore to evaluate the two separate production lots of a commercial POC lateral flow test, intended for the detection of IgM and IgG against the SARS-CoV-2 spike protein (S1). Control samples consisted of serum from individuals with confirmed SARS-CoV-2 infection and pre-COVID-19 negative sera gathered from a biobank. The presence of anti-S1 IgM/IgG in the sera was verified by an in-house Luminex-based serological assay (COVID-19 SIA). One hundred samples were verified as positive for anti-S1 IgG and 74 for anti-S1 IgM. Two hundred samples were verified as negative for anti-S1 IgM/IgG. For the two lots of the POC-test, the sensitivities were 93.2% and 87.8% for IgM and 93.0% and 100% for IgG. The specificities were 100% for IgM and 99.5% for IgG. The positive predictive value was 100% for IgM and 98.9% and 99.0% for IgG. The negative predictive value was 97.6% and 95.7% for IgM, and 96.6% and 100% for IgG. The evaluated POC-test is suitable to assess anti-SARS-CoV-2 S1 IgM and IgG, as a measure of previous virus exposure on an individual level. The external validation of separate lots of rapid POC-tests is encouraged to ensure high sensitivity before market introduction.

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750 Malat1 lncRNA controls metastatic reactivation of dormant breast cancer by immune evasion

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0750 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A799-A799

Author(s):

Dhiraj Kumar ◽

Sreeharsha Gurrapu ◽

Hyunho Han ◽

Yan Wang ◽

Seongyeon Bae ◽

...

Keyword(s):

Breast Cancer ◽

T Cells ◽

Single Cell ◽

Cancer Cells ◽

Tumor Cells ◽

Immune Evasion ◽

Breast Cancer Cells ◽

Metastatic Potential ◽

Rna Seq

BackgroundLong non-coding RNAs (lncRNAs) are involved in various biological processes and diseases. Malat1 (metastasis-associated lung adenocarcinoma transcript 1), also known as Neat2, is one of the most abundant and highly conserved nuclear lncRNAs. Several studies have shown that the expression of lncRNA Malat1 is associated with metastasis and serving as a predictive marker for various tumor progression. Metastatic relapse often develops years after primary tumor removal as a result of disseminated tumor cells undergoing a period of latency in the target organ.1–4 However, the correlation of tumor intrinsic lncRNA in regulation of tumor dormancy and immune evasion is largely unknown.MethodsUsing an in vivo screening platform for the isolation of genetic entities involved in either dormancy or reactivation of breast cancer tumor cells, we have identified Malat1 as a positive mediator of metastatic reactivation. To functionally uncover the role of Malat1 in metastatic reactivation, we have developed a knock out (KO) model by using paired gRNA CRISPR-Cas9 deletion approach in metastatic breast and other cancer types, including lung, colon and melanoma. As proof of concept we also used inducible knockdown system under in vivo models. To delineate the immune micro-environment, we have used 10X genomics single cell RNA-seq, ChIRP-seq, multi-color flowcytometry, RNA-FISH and immunofluorescence.ResultsOur results reveal that the deletion of Malat1 abrogates the tumorigenic and metastatic potential of these tumors and supports long-term survival without affecting their ploidy, proliferation, and nuclear speckles formation. In contrast, overexpression of Malat1 leads to metastatic reactivation of dormant breast cancer cells. Moreover, the loss of Malat1 in metastatic cells induces dormancy features and inhibits cancer stemness. Our RNA-seq and ChIRP-seq data indicate that Malat1 KO downregulates several immune evasion and stemness associated genes. Strikingly, Malat1 KO cells exhibit metastatic outgrowth when injected in T cells defective mice. Our single-cell RNA-seq cluster analysis and multi-color flow cytometry data show a greater proportion of T cells and reduce Neutrophils infiltration in KO mice which indicate that the immune microenvironment playing an important role in Malat1-dependent immune evasion. Mechanistically, loss of Malat1 is associated with reduced expression of Serpinb6b, which protects the tumor cells from cytotoxic killing by the T cells. Indeed, overexpression of Serpinb6b rescued the metastatic potential of Malat1 KO cells by protecting against cytotoxic T cells.ConclusionsCollectively, our data indicate that targeting this novel cancer-cell-initiated domino effect within the immune system represents a new strategy to inhibit tumor metastatic reactivation.Trial RegistrationN/AEthics ApprovalFor all the animal studies in the present study, the study protocols were approved by the Institutional Animal Care and Use Committee(IACUC) of UT MD Anderson Cancer Center.ConsentN/AReferencesArun G, Diermeier S, Akerman M, et al., Differentiation of mammary tumors and reduction in metastasis upon Malat1 lncRNA loss. Genes Dev 2016 Jan 1;30(1):34–51.Filippo G. Giancotti, mechanisms governing metastatic dormancy and reactivation. Cell 2013 Nov 7;155(4):750–764.Gao H, Chakraborty G, Lee-Lim AP, et al., The BMP inhibitor Coco reactivates breast cancer cells at lung metastatic sites. Cell 2012b;150:764–779.Gao H, Chakraborty G, Lee-Lim AP, et al., Forward genetic screens in mice uncover mediators and suppressors of metastatic reactivation. Proc Natl Acad Sci U S A 2014 Nov 18; 111(46): 16532–16537.

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Metastatic Esophageal Carcinoma Cells Exhibit Reduced Adhesion Strength and Enhanced Thermogenesis

Cells ◽

10.3390/cells10051213 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1213

Author(s):

Zihe Huo ◽

Mariana Sá Santos ◽

Astrid Drenckhan ◽

Stefan Holland-Cunz ◽

Jakob R. Izbicki ◽

...

Keyword(s):

Esophageal Carcinoma ◽

Tumor Cells ◽

Cell Lines ◽

Adhesion Strength ◽

Primary Tumor ◽

Carcinoma Cell ◽

Metastatic Potential ◽

Primary Tumors ◽

Metastatic Cell ◽

Carcinoma Cell Lines

Despite continuous improvements in multimodal therapeutic strategies, esophageal carcinoma maintains a high mortality rate. Metastases are a major life-limiting component; however, very little is known about why some tumors have high metastatic potential and others not. In this study, we investigated thermogenic activity and adhesion strength of primary tumor cells and corresponding metastatic cell lines derived from two patients with metastatic adenocarcinoma of the esophagus. We hypothesized that the increased metastatic potential of the metastatic cell lines correlates with higher thermogenic activity and decreased adhesion strength. Our data show that patient-derived metastatic esophageal tumor cells have a higher thermogenic profile as well as a decreased adhesion strength compared to their corresponding primary tumor cells. Using two paired esophageal carcinoma cell lines of primary tumor and lymph nodes makes the data unique. Both higher specific thermogenesis profile and decreased adhesion strength are associated with a higher metastatic potential. They are in congruence with the clinical patient presentation. Understanding these functional, biophysical properties of patient derived esophageal carcinoma cell lines will enable us to gain further insight into the mechanisms of metastatic potential of primary tumors and metastases. Microcalorimetric evaluation will furthermore allow for rapid assessment of new treatment options for primary tumor and metastases aimed at decreasing the metastatic potential.

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