Single-cell atlas of tumor cell evolution in response to therapy in hepatocellular carcinoma and intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: Intratumor molecular heterogeneity is a key feature of tumorigenesis and is linked to treatment failure and patient prognosis. Herein, we aimed to determine what drives tumor cell evolution by performing single-cell transcriptomic analysis. METHODS: We analyzed 46 hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA) biopsies from 37 patients enrolled in interventional studies at the NIH Clinical Center, with 16 biopsies collected before and after treatment from 7 patients. We developed a novel machine learning-based consensus clustering approach to track cellular states of 57,000 malignant and non-malignant cells including tumor cell transcriptome-based functional clonality analysis. We determined tumor cell relationships using RNA velocity and reverse graph embedding. We also studied longitudinal samples from 4 patients to determine tumor cellular state and its evolution. We validated our findings in bulk transcriptomic data from 488 patients with HCC and 277 patients with iCCA. RESULTS: Using transcriptomic clusters as a surrogate for functional clonality, we observed an increase in tumor cell state heterogeneity which was tightly linked to patient prognosis. Furthermore, increased functional clonality was accompanied by a polarized immune cell landscape which included an increase in pre-exhausted T cells. We found that SPP1 expression was tightly associated with tumor cell evolution and microenvironmental reprogramming. Finally, we developed a user-friendly online interface as a knowledge base for a single-cell atlas of liver cancer. CONCLUSIONS: Our study offers insight into the collective behavior of tumor cell communities in liver cancer as well as potential drivers of tumor evolution in response to therapy. LAY SUMMARY: Intratumor molecular heterogeneity is a key feature of tumorigenesis that is linked to treatment failure and patient prognosis. In this study, we present a single-cell atlas of liver tumors from patients treated with immunotherapy and describe intratumoral cell states and their hierarchical relationship. We suggest osteopontin, encoded by the gene SPP1, as a candidate regulator of tumor evolution in response to treatment.

Multifaceted modifications for a cell size-based circulating tumor cell scope technique hold the prospect for large-scale application in general populations.

Circulating tumor cells (CTCs) indicate the diagnosis and prognosis of cancer patients, together with benefiting individual treatment and anticancer drug development. However, their large-scale application in general population still requires systematically multifaceted modifications for currently proprietary new technologies based on filtration. We primitively utilized a cell size-based platform to evaluate the recovery efficiency of spiked abnormal cell lines and analyzed circulating abnormal cells (CACs). To dissect the subpopulations of CACs, we conducted immunofluorescent (IF) staining with a combination of unique biomarkers of CTCs and circulating endothelial cells (CECs). Furthermore, we improved the CTC screening system by assessing the feasibility of transferring CTCs for automatic IF analysis, together with simulating and optimizing the circumstances for long-term CTC storage and transportation. We detected CACs in 15 HD candidates with CTC characteristics such as abnormally large cytomorphology, high nuclear-cytoplasmic ratio, and positive for panCK or VIM staining. Thereafter, we improved accuracy of the platform by distinguishing CTCs from CECs, which satisfied the elementary requirement for small-scale CTC screening in HD candidates. Finally, large-scale CTC screening in general population was available after multifaceted modifications including automatic analysis by transferring CTCs on slides, choosing the appropriate blood-collecting tube, optimizing the conditions for long-term CTC storage and transportation, and evaluating the potential effect on the CTC phenotype. Hence, we systematically modified the scope of technique parameters, improved the accuracy of early cancer detection, and made it realizable for large-scale CTC or CEC screening in general population.

Binary-blend fibber-based capture assay of circulating tumor cells for clinical diagnosis of colorectal cancer.

BACKGROUND: In addition to conventional approaches, detecting and characterizing CTCs in patient blood allows for early diagnosis of cancer metastasis. METHODS: We blended poly(ethylene oxide) (PEO) into nylon-6 through electrospinning to generate a fibrous matbased circulating tumour cells (CTCs) assay. The contents of nylon-6 and PEO in the electrospun blend fibrous mats (EBFMs) were optimized to facilitate high cell-substrate affinity and low leukocyte adsorption. RESULTS: Compared with the IsoFlux System, a commercial instrument for CTC detection, the CTC assay of EBFMs exhibited lower false positive readings and high sensitivity and selectivity with preclinical specimens. Furthermore, we examined the clinical diagnosis accuracy of colorectal cancer, using the CTC assay and compared the results with those identified through pathological analyses of biopsies from colonoscopies. Our positive expressions of colorectal cancer through CTC detection completely matched those recognized through the pathological analyses for the individuals having stage II, III, and IV colorectal cancer. Nevertheless, two in four individuals having stage I colorectal cancer, recognized through pathological analysis of biopsies from colonoscopies, exhibited positive expression of CTCs. Ten individuals were identified through pathological analysis as having no colorectal tumours. Nevertheless, two of these ten individuals exhibited positive expression of CTCs. CONCLUSIONS: Thus, in this population, the low cost EBFMs exhibited considerable capture efficiency for the non-invasive diagnosis of colorectal cancer.

Proportion of circulating tumor cell clusters increases during cancer metastasis.

Circulating tumor cell (CTC) clusters are found among CTCs and show significantly greater potential for an important role in cancer metastasis than single CTCs, which have been traditionally believed as the majority of CTCs. The accurate proportion and dynamics of CTC clusters remain unclear due to the fact that CTCs in blood flow are very difficult to detect in vivo and in vitro. CTC clusters are even more difficult to be distinguished from CTCs without perturbation by state-of-the-art detection methods. Here, we demonstrate that by using in vivo flow cytometry (IVFC), we can reliably measure the proportion and dynamics of CTC clusters in two murine tumor models. CTC clusters are easily identified by their unique fluorescent pattern with multiple peaks and wider time duration. We find that the proportion of CTC clusters increases significantly during cancer metastasis in both mouse models, the orthotopic liver cancer and the subcutaneous prostate cancer models. Our results suggest that CTC clusters account for a much larger proportion of CTCs than previously anticipated. Hence this report might provide a new-level of understanding of CTCs during cancer development and progression. © 2016 International Society for Advancement of Cytometry.

Automated detection of circulating tumor cells with naive Bayesian classifiers.

Personalized medicine is a modern healthcare approach where information on each person's unique clinical constitution is exploited to realize early disease intervention based on more informed medical decisions. The application of diagnostic tools in combination with measurement evaluation that can be performed in a reliable and automated fashion plays a key role in this context. As the progression of various cancer diseases and the effectiveness of their treatments are related to a varying number of tumor cells that circulate in blood, the determination of their extremely low numbers by liquid biopsy is a decisive prognostic marker. To detect and enumerate circulating tumor cells (CTCs) in a reliable and automated fashion, we apply methods from machine learning using a naive Bayesian classifier (NBC) based on a probabilistic generative mixture model. Cells are collected with a functionalized medical wire and are stained for fluorescence microscopy so that their color signature can be used for classification through the construction of Red-Green-Blue (RGB) color histograms. Exploiting the information on the fluorescence signature of CTCs by the NBC does not only allow going beyond previous approaches but also provides a method of unsupervised learning that is required for unlabeled training data. A quantitative comparison with a state-of-the-art support vector machine, which requires labeled data, demonstrates the competitiveness of the NBC method.

Primitive neuroectodermal tumors of adrenal gland.

OBJECTIVES: To analyze the clinical and pathological characteristics of adrenal primitive neuroectodermal tumors for a better understanding of the disease. METHODS: A retrospective analysis of four cases of adrenal primitive neuroectodermal tumors (two male, two female; age 21-30, average 24) was made. All patients went through necessary endocrinological exams, computer tomography scans (for site-specific diagnoses) and pathological tests. RESULTS: No positive result was reported in routine laboratory tests and endocrinological exams. Computer tomography scans showed bulk soft tissue masses with rough borders. The masses were 8-17 cm in diameter with solid-cystic changes. Among the four patients, one refused to receive treatment after definitiver diagnosis through needle biopsy, three received surgical treatments and their post-operative pathological exams all confirmed the diagnoses of primitive neuroectodermal tumors. During the follow-ups, the untreated patient died 6 months afterwards, one patient died 8 months after palliative treatment; one patient showed distant metastasis 13 months after surgery and did not respond well to both radio- and chemotherapy; one patient had local recurrence 1 month after surgery and is presently undergoing chemotherapy. CONCLUSIONS: Adrenal primitive neuroectodermal tumor is a very rare tumor. It originates in primitive neuroectoderma and is found mainly in 20-30-year-old young populations. It has non-specific clinical or imaging manifestation and its diagnosis is mostly based on pathological examinations. The tumor is fast-developing, highly malignant with poor prognosis.

Photodynamic inactivation of circulating tumor cells: An innovative approach against metastatic cancer.

The spread of a primary malignant tumor is the major reason for most of the cancer-associated deaths. To this day, treatment regimen and available drugs are still insufficient to manage these conditions. In this work, a new therapeutic concept based on photodynamic therapy (PDT) of metastasis-initiating cells is introduced. To address this issue, an experimental model was developed to simulate the movement and photodynamic inactivation of circulating tumor cells (CTCs) in vitro. Using curcumin loaded poly(lactic-co-glycolic acid) nanoparticles, a significant reduction in the cell viability of human breast cancer cells (MDA-MB-231) could be achieved after 30 min laser irradiation (λ = 447 nm, P = 100mW) under flow conditions (5 cm s-1). Confocal laser scanning microscopy images confirmed the immediate accumulation of curcumin on the cell membrane and an increased fluorescence signal after irradiation. PDT caused time-dependent morphological cell alterations (i.e. membrane evaginations and disruption) indicating apoptosis and early necrosis. During the photoactivation of curcumin, a blue shift in the absorption spectra and a decrease in the curcumin content could be determined. This study confirms that the presented experimental model is suitable for in vitro investigations of CTCs under in vivo-like conditions, at the same time encouraging the clinical implementation of PDT as an innovative strategy against metastasis.

Light-driven ultrasensitive self-powered cytosensing of circulating tumor cells via integration of biofuel cells and a photoelectrochemical strategy.

Herein, a light-driven, membrane-less and mediator-less self-powered cytosensing platform via integration of biofuel cells (BFCs) and a photoelectrochemical strategy was developed for ultrasensitive detection of circulating tumor cells (CTCs). To construct cytosensors, an elaborately designed SH-Sgc8c aptamer/AuNP/g-C3N4 photoelectrode was used as an alternative anode for glucose oxidation, avoiding the introduction of anodic enzymes. Initially, glucose could favorably reach the photoanode surface and be easily oxidized by the photogenerated holes, while the photogenerated electrons would transfer to the biocathode and achieve biocatalytic reduction of O2, leading to a high EOCV. However, in the presence of CTCs, they could preferentially interact with the Sgc8c aptamer via specific recognition, and then complexes with large steric hindrance were immobilized on the photoanode surface, which could greatly affect the electron transfer between glucose and the photoanode surface. In this case, the EOCV decreased sharply. Encouragingly, this self-powered cytosensor exhibited an ultrasensitive response to the target CTCs in a wide concentration range from 20 to 2 × 105 cells mL-1 with a low detection limit of 10 cells mL-1 (S/N = 3), being superior to those of the reported methods. Moreover, this as-proposed self-powered cytosensor integrated with a photoelectrochemical strategy possessed unique advantages of not requiring an external power source, being anodic enzyme-free, having a simple construction process, facile miniaturization, and high selectivity and sensitivity, providing a promising and powerful tool for fundamental biochemical research and clinical diagnosis.

Clinical Potential of Circulating Tumor Cells in Colorectal Cancer: A Prospective Study.

OBJECTIVES: Circulating tumor cells (CTCs) in the blood have been used as diagnostic markers in patients with colorectal cancer (CRC). In this study, we evaluated a CTC detection system based on cell size to assess CTCs and their potential as early diagnostic and prognostic biomarkers for CRC. METHODS: From 2014 to 2015, 88 patients with newly diagnosed CRC, who were scheduled for surgery, and 31 healthy volunteers were enrolled and followed up in Pusan National University Hospital. CTCs were enriched using a centrifugal microfluidic system with a new fluid-assisted separation technique (FAST) and detected by cytomorphological evaluation using fluorescence microscopy. RESULTS: Two or more CTCs were detected using FAST in 74 patients and 3 healthy volunteers. The number of CTCs in the CRC group was significantly higher than that in the healthy volunteers (P < 0.001). When a receiver operating characteristic curve was created to differentiate patients with CRC from healthy volunteers, the sensitivity and specificity were almost optimized when the critical CTC value was 5/7.5 mL of blood. When this value was used, the sensitivity and specificity in differentiating patients with CRC from the healthy controls were 75% and 100%, respectively. In patients with CRC with ≥5 CTCs, vascular invasion was frequently identified (P = 0.035). All patients with stage IV were positive for CTCs. Patients with ≥5 CTCs showed a trend toward poor overall and progression-free survival. DISCUSSION: Our study demonstrated promising results with the use of FAST-based CTC detection for the early diagnosis and prognosis of CRC.

Membrane microfilter device for selective capture, electrolysis and genomic analysis of human circulating tumor cells.

This paper presents development of a parylene membrane microfilter device for single stage capture and electrolysis of circulating tumor cells (CTCs) in human blood, and the potential of this device to allow genomic analysis. The presence and number of CTCs in blood has recently been demonstrated to provide significant prognostic information for patients with metastatic breast cancer. While finding as few as five CTCs in about 7.5mL of blood (i.e., 10(10) blood cells in) is clinically significant, detection of CTCs is currently difficult and time consuming. CTC enrichment is performed by either gradient centrifugation of CTC based on their buoyant density or magnetic separation of epithelial CTC, both of which are laborious procedures with variable efficiency, and CTC identification is typically done by trained pathologists through visual observation of stained cytokeratin-positive epithelial CTC. These processes may take hours, if not days. Work presented here provides a micro-electro-mechanical system (MEMS)-based option to make this process simpler, faster, better and cheaper. We exploited the size difference between CTCs and human blood cells to achieve the CTC capture on filter with approximately 90% recovery within 10 min, which is superior to current approaches. Following capture, we facilitated polymerase chain reaction (PCR)-based genomic analysis by performing on-membrane electrolysis with embedded electrodes reaching each of the individual 16,000 filtering pores. The biggest advantage for this on-membrane in situ cell lysis is the high efficiency since cells are immobilized, allowing their direct contact with electrodes. As a proof-of-principle, we show beta actin gene PCR, the same technology can be easily extended to real time PCR for CTC-specific transcript to allow molecular identification of CTC and their further characterization.

Cryopreservation of Circulating Tumor Cells for Enumeration and Characterization.

BACKGROUND: A blood sample containing circulating tumor cells (CTCs) may serve as a surrogate for metastasis in invasive cancer. Cryopreservation will provide new opportunities in management of clinical samples in the laboratory and allow collection of samples over time for future analysis of existing and upcoming cancer biomarkers. METHODS: Blood samples from healthy volunteers were spiked with high (∼500) and low (∼50) number of tumor cells from culture. The samples were stored at -80C with cryopreservative dimethyl sulfoxide mixed with Roswell Park Memorial Institute 1640 medium. Flow cytometry tested if cryopreservation affected specific biomarkers regularly used to detect CTCs, i.e. cytokeratin (CK) and epithelial cell adhesion molecule (EpCAM) and white blood cell specific lymphocyte common antigen (CD45). After various time intervals (up to 6 months), samples were thawed and tumor cell recovery (enumeration) was examined. Clinical samples may differ from cell line studies, so the cryopreservation protocol was tested on 17 patients with invasive breast cancer and tumor cell recovery was examined. Two blood samples were drawn from each patient. RESULTS: Biomarkers, CK, CD45, and EpCAM, were not affected by the freezing and thawing procedures. Cryopreserved samples (n = 2) spiked with a high number of tumor cells (∼500) had a ∼90% recovery compared with the spiked fresh samples. In samples spiked with lower numbers of tumor cells (median = 43 in n = 5 samples), the recovery was 63% after cryopreservation (median 27 tumor cells), p = 0.03. With an even lower number of spiked tumor cells (median = 3 in n = 8 samples), the recovery rate of tumor cells after cryopreservation did not seem to be affected (median = 8), p = 0.09. Time of cryopreservation did not affect recovery. When testing the effect of cryopreservation on enumeration in clinical samples, no difference was observed in the number of CTCs between the fresh and the cryopreserved samples based on n = 17 pairs, p = 0.83; however, the variation was large. This large variation was confirmed by clinically paired fresh samples (n = 64 pairs), where 95% of the samples (<30 CTCs) vary in number up to ±15 CTCs, p = 0.18. CONCLUSIONS: A small loss of CTCs after cryopreservation may be expected; however, cryopreservation of CTCs for biomarker characterization for clinical applications seems promising.

Adhesion receptors on peripheral blood leukemic B cells. A comparative study on B cell chronic lymphocytic leukemia and related lymphoma/leukemias.

The expression of a series of adhesion receptors: L-selectins (CD62L): Leu-8, several integrins (LFA-1: CD11a/CD18, VLA-4: CD49d/CD29 and VLA-5: CD49e/CD29), ICAM-1(CD54) and the 'homing receptor' (CD44) were investigated by a dual color flow cytometry in 56 cases of B cell disorders namely, 39 chronic lymphocytic leukemias (CLL), four hairy cell leukemia (HCL), seven splenic lymphoma with villous lymphocytes (SLVL) and six other non-Hodgkin's lymphoma (NHL). The functional activity of L-selectins was assessed with L-selectin ligand analogs (polyphosphomonester core polysaccharide: PPME and fucoidin). Leukemic B cells were identified with phycoerythrin-conjugated monoclonal antibodies (McAbs) anti-CD19, anti-kappa/lambda investigated simultaneously for the expression of adhesion receptors estimated with fluorescein-isothiocyanate (FITC) conjugated McAbs. The percentage of leukemic cells expressing L-selectins (Leu-8) was high in CLL (52% of positive cases) and integrin expression (LFA-1, VLA-4, 5) was low (19 and 33%, respectively), while a reverse pattern, low Leu-8 (17%), and a high VLA-4 (77%), was observed in non-CLL cases. The expression of LFA-1 alpha-chain was variable in non-CLL cases, and the LFA-1 heterodimer was expressed on most clonal B cell in NHLs (92%). LFA-1 alpha-chain was detected on cells from only one HCL case, while beta2 integrin was regularly expressed on hairy cells. VLA-5 integrin was found on a relatively small number (26%) of mature B cell leukemias. A remarkable finding was the detection of ICAM-1 in all CLL cases albeit the number of positive cells was significantly lower (P < 0.05) compared to non-CLL cases. CD44 was expressed on a high number of neoplastic cells in all the investigated categories. There was no correlation between the expression of the adhesion molecules and clinical and laboratory parameters except for CD18 which was expressed on a significantly (P < 0.05) higher number of leukemic cells in CLL with more advanced stages. This study demonstrates that even closely related B cell leukemia/lymphomas have a certain well defined and strictly variable adhesion profile which is characteristic of the disease entity and therefore, the adhesion profile may offer additional information useful for differential diagnosis and study of disease pathogenesis.

Realtime visualization of tumor cell/endothelial cell interactions during transmigration across the endothelial barrier.

PURPOSE: In cancer the blood-borne spread of tumor cells leads to the formation of secondary tumors at distant loci whereby the extravasation of tumor cells is a prerequisite step during hematogenous metastasis. Here, we describe a novel in vitro realtime model which shows the complete sequence of the extravasation process. METHODS: We developed an in vitro system allowing us to monitor the sequence of extravasation events of tumor cell clusters across a monolayer of human umbilical cord endothelial cells (HUVEC). Fluorescence markers and laser scanning confocal microscopy were used to visualize the interactions between tumor cells and endothelium. RESULTS: Our model indicates that the extravasation of tumor cell clusters derived from the invasive human bladder carcinoma cell line T24 occurs in a relatively short time-frame up to 4 h after adhesion to the endothelium. We demonstrate that the vascular endothelium is irreversibly damaged at the site of tumor cell extravasation. CONCLUSION: Realtime laser scanning confocal microscopy leads to a better understanding of the complex and dynamic cell-to-cell and cell-to-matrix interactions during the extravasation process.

Auer rod-like inclusions in circulating lymphoma cells.

Circulating malignant lymphocytes from a 55-year-old woman with small cleaved follicular center cell lymphoma contained azurophilic splinter-shaped cytoplasmic inclusions. By light microscopic and ultrastructural criteria, these structures closely resembled Auer rods found in acute myeloid leukemia; however, the authors could not find cytochemical evidence of lysosomal origin (results were negative for myeloperoxidase, Sudan black B, acid phosphatase, and periodic acid-Schiff). Immunostaining and flow cytometric analysis confirmed a monoclonal IgM-kappa immunophenotype of the circulating malignant lymphoid cells. The inclusions did not show specific immunoglobulin staining by light microscopic or electron microscopic immunostaining techniques. The authors conclude that these membrane-bound inclusions probably represent aberrant lysosomes in the malignant cells.

Splenic lymphoma with circulating villous lymphocytes. Report of a case with immunologic and ultrastructural studies.

A case of splenic lymphoma with circulating villous lymphocytes is reported. Short surface cellular expansions were observed on blood and marrow films and by transmission electron microscopy. The immunophenotype was that of mature B cells without CD5, CD10, CD11c, or CD25 expression or tartrate-resistant acid phosphatase. Despite a basophilic plasmacytoid-like cytoplasm, this case of splenic lymphoma with circulating villous lymphocytes differed from splenic immunocytoma in that immunofluorescence and ultrastructure suggested that the neoplastic cells did not possess high levels of intracytoplasmic immunoglobulin. Treatment of cytopenia was best achieved by splenectomy and the total follow-up thus far (30 months) seems to indicate a case of low-grade malignant lymphoma.

Critical steps in hematogenous metastasis: an overview.

Metastasis is responsible for most cancer deaths. A better understanding of the process provides opportunities to develop new treatments to prevent metastasis. This article summarizes findings from experimental in vivo videomicroscopy and quantitative studies on metastatic inefficiency, which indicate that early steps in hematogenous metastasis may be quite efficient, but that regulation of cancer cell growth in secondary sites determines metastatic outcome. The authors have identified three key stages of this growth regulation: survival of a subset of single cells, proliferation of a subset of these cells to form preangiogenic micrometastases, and persistence of growth of a subset of these to form vascularized metastases. Formation of clinically relevant metastases is determined by the proportion of cells that proceeds successfully through each stage, and surviving single cells and preangiogenic micrometastases both represent possible sources of tumor dormancy.

To the incidence of nucleoli in circulating myeloblasts of patients suffering from acute myeloblastic, promyelocytic and myelomonocytic leukemias.

Nucleoli were studied in circulating myeloblasts of myeloblastic (FAB M1, M2), promyelocytic (FAB M3) and myelomonocytic (FAB M4) acute myeloid leukemias (AMLs) using a cytochemical procedure for the demonstration of RNA. In patients untreated with cytostatic chemotherapy, myeloblasts of myeloblastic acute leukemias possessed less frequently "active large" nucleoli and more frequently "inactive" micronucleoli in comparison with other investigated types of AMLs. When myeloblasts were classified according to the presence of functionally dominant nucleoli, the higher percentage of "terminal" myeloblasts containing only micronucleoli in this type of AML was significantly reduced in patients treated with the cytostatic chemotherapy. In patients suffering from promyelocytic leukemia treated with cytostatic chemotherapy, the decreased percentage of myeloblasts containing functionally dominant active large nucleoli was accompanied by the increased incidence of myeloblasts with functionally dominant "resting" ring shaped nucleoli. In myelomonocytic AML no significant differences were noted between patients untreated or treated with the cytostatic chemotherapy in the incidence of main nucleolar types in myeloblasts and myeloblasts classified according to the presence of functionally dominant nucleoli. Thus a further biological specificity might exist among leukemic blasts in various types of AMLs which should be considered for a rational approach to the therapy of these malignancies. In addition, the cytostatic chemotherapy did not influence incidence of the nucleolar asynchrony in myeloblasts of all investigated types of AML.

Morphological differences between circulating tumor cells from prostate cancer patients and cultured prostate cancer cells.

Circulating tumor cell (CTC) enumeration promises to be an important predictor of clinical outcome for a range of cancers. Established CTC enumeration methods primarily rely on affinity capture of cell surface antigens, and have been criticized for underestimation of CTC numbers due to antigenic bias. Emerging CTC capture strategies typically distinguish these cells based on their assumed biomechanical characteristics, which are often validated using cultured cancer cells. In this study, we developed a software tool to investigate the morphological properties of CTCs from patients with castrate resistant prostate cancer and cultured prostate cancer cells in order to establish whether the latter is an appropriate model for the former. We isolated both CTCs and cultured cancer cells from whole blood using the CellSearch® system and examined various cytomorphological characteristics. In contrast with cultured cancer cells, CTCs enriched by CellSearch® system were found to have significantly smaller size, larger nuclear-cytoplasmic ratio, and more elongated shape. These CTCs were also found to exhibit significantly more variability than cultured cancer cells in nuclear-cytoplasmic ratio and shape profile.

Live cell imaging in live animals with fluorescent proteins.

The discovery, cloning, and characterization of GFP and related proteins of many colors have enabled live cell imaging to an unprecedented extent and resolution. Essentially, any cellular process can be imaged with a fluorescent protein. These proteins serve as genetic reporters and therefore can be used to follow cellular processes over indefinite periods in vivo as well as in vitro. The brightness and specific spectra of fluorescent proteins allow them to be imaged in vivo, using specific filters, without interference from autofluorescence. This chapter describes the development of live imaging in live animals with subcellular resolution, emphasizing the study of in vivo cell biology of cancer growth, spread, and metastasis.

Shear stress induces not only platelet aggregation but also platelet-tumor cell interaction.

To investigate the interaction between platelets and tumor cells under well-defined flow conditions, the effect of tumor cells on platelet aggregation induced by shear stress was studied using a cone and plate viscometer adapted for measuring transmitted light intensity. Aggregation was markedly enhanced by HMV-1 cells in a cell number-dependent fashion under shear stress of 12 dyne/cm2. Enhancement was not observed at a high shear stress of 108 dyne/cm2. A monoclonal antibody against GPIIb/IIIa, 7E3 completely abolished enhancement of aggregation by HMV-1. Apyrase had similar inhibitory effects. Scanning electronmicroscopy showed that direct contacts of platelets with HMV-1 cells could be demonstrated when platelet-platelet interaction was inhibited by 7E3 or apyrase. These results may indicate that, at a shear stress of 12 dyne/cm2, direct contacts of platelets and HMV-1 cells may trigger enhancement of platelet aggregation.