Cancer Cells In Blood Samples Research Articles

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.

Abstract 6605: Automated isolation and identification of circulating rare cells from blood samples using a label-free rare cell isolation platform - the Genesis System with Celselect Slide technology

Abstract Introduction: Isolation and identification of circulating tumor cells (CTCs) offers a promising non-invasive diagnostic tool for cancer research, but significant technical hurdles have made practical application challenging. Recent studies have shown that CTCs undergo cellular changes, such as an epithelial-mesenchymal transition (EMT), with disease progression and metastasis, implying the heterogeneity of the tumor cell population in circulation. Systems for CTC isolation are often limited by a requirement for epithelial markers or separate systems for identification of CTCs. As such, an automated platform that successfully isolates both epithelial and mesenchymal cells and enables identification of cells is necessary for successful study of CTCs. The Genesis System from Bio-Rad™ Laboratories, Inc. isolates rare CTCs based on size and deformability. It also enables users to identify isolated cells through use of an on-slide immunofluorescence (IF) staining workflow, which minimizes sample manipulation and potential sample loss. Methods: In order to achieve high-throughput label-free CTC isolation, the Genesis System uses Celselect Slides™. Each slide contains 56,400 individual microchambers to capture CTCs while maintaining viability. This Celselect Slide technology can be used for either enrichment or enumeration protocols. For this study, enumeration protocols were performed to capture and identify CTCs using on-slide IF staining. Enrichment protocols were utilized to capture, enrich, and retrieve CTCs for downstream applications. The performance was evaluated using a breast cancer cell line, MCF-7, spiked into whole blood from healthy donors. Results: The slide isolated 83.95 ± 10.25% of spiked cancer cells in blood samples at a concentration of 10-250 cells/ml. During the CTC enrichment procedure, 99.92% of blood cells were eliminated. Afterwards, 70% of the isolated CTCs were successfully released from the slide and over 92% of the released cells remained viable. Discussion and Conclusion: We present a fully automated platform to isolate and identify CTCs using on-slide immunofluorescence staining, which minimizes inevitable cell loss during the workflow. Captured cells can be retrieved for downstream applications to facilitate discovery of unknown CTC functions and their clinical relevance in cancer research. The wide range of possible downstream applications include Droplet Digital™ PCR, quantitative PCR, western blot assays, bulk/single-cell genomics, cell culture, fluorescence in situ hybridization, and RNA-Seq. Additionally, the design of the system is amenable to other biofluids which may be utilized for liquid biopsies. The Genesis System with Celselect Slide technology offers a powerful diagnostic tool to monitor disease progression and personalized treatment. Citation Format: Yoon-Tae Kang, Errile Pusod, Ally Crudgington, Abiodun Bodunrin, Nick Bosio, Susana Chan, David Coe, Michelle Racey. Automated isolation and identification of circulating rare cells from blood samples using a label-free rare cell isolation platform - the Genesis System with Celselect Slide technology. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6605.

Multimodal CTC detection using stem cell antigen-specific immunosilica particles and immunofluorescent quantum dots

Amid the COVID-19 pandemic, cancer continues to be the most devastating disease worldwide. Liquid biopsy of circulating tumor cells (CTCs) has recently become a painless and noninvasive tool for obtaining carcinoma cell samples for molecular profiling. Here, we report efficient detection and collection of cancer cells in blood samples by combining stem cell antigen (CD44)-specific immunosilica particles and immunofluorescent quantum dots with spectrally and temporally resolved single-photon counting. We accurately detect 1–10 cells among 100 cancer cells of the breast, lungs, or cervix in 1 mL blood samples. In addition, the bright and narrowband emission of CdSe/ZnS quantum dots enables temporally and spectrally resolved photon counting for multiplexed cancer cell detection. The cancer cell-specific and large immunosilica particles helped us collect the specific cells. We validate the detection efficiency and multimodality of this strategy by time-stamped and energy-dispersed single-photon counting of orange- and red-emitting quantum dots and green-fluorescing nuclei stained with Syto-13/25 dye. Thus, the present work highlights the prospects of multimodal CTC detection for noninvasive cancer screening and postsurgical or therapeutic follow-up.

High-recovery sorting of cancer cells from whole blood via periodic-focusing inertial microchip.

Inertial microfluidic devices continue to show promise for label-free separation of cells from liquid biopsies and other biological samples. Serpentine-channel microfluidic devices capitalizing on inertial forces such as Dean flow have been demonstrated for cell separation, but are limited in performance due to the magnitude of the inertial lift and drag force gradients across the separation channel. We have developed a new flow design that uses periodic channel contractions to enhance the magnitude of the force gradient. Separation recover was 97% with the final sorter output consisting of 78% target cells. Separation efficiency was 87% for whole blood, which could be increased to 97% if the sample was diluted prior to sorting. The enrichment of cancer cells was over 1000-fold, and sorted cancer cells maintained a viability of 93.8% for 96 hours after sorting. In the analysis of blood plasma, breast cancer cells from a clinical patient were enriched 20×. The incorporation of periodic channel contractions in a Dean flow circuit resulted in an increase in Dean flow gradient according to simulation, resulting in sorting of small-diameter cancer cells in blood samples.

Cancer cell enrichment on a centrifugal microfluidic platform using hydrodynamic and magnetophoretic techniques

Isolation of rare cancer cells is one of the important and valuable stages of cancer research. Regarding the rarity of cancer cells in blood samples, it is important to invent an efficient separation device for cell enrichment. In this study, two centrifugal microfluidic devices were designed and fabricated for the isolation of rare cancer cells. The first design (passive plan) employs a contraction–expansion array (CEA) microchannel which is connected to a bifurcation region. This device is able to isolate the target cells through inertial effects and bifurcation law. The second design (hybrid plan) also utilizes a CEA microchannel, but instead of using the bifurcation region, it is reinforced by a stack of two permanent magnets to capture the magnetically labeled target cells at the end of the microchannel. These designs were optimized by numerical simulations and tested experimentally for isolation of MCF-7 human breast cancer cells from the population of mouse fibroblast L929 cells. In order to use the hybrid design, magnetite nanoparticles were attached to the MCF-7 cells through specific Ep-CAM antibodies, and two permanent magnets of 0.34 T were utilized at the downstream of the CEA microchannel. These devices were tested at different disk rotational speeds and it was found that the passive design can isolate MCF-7 cells with a recovery rate of 76% for the rotational speed of 2100 rpm while its hybrid counterpart is able to separate the target cells with a recovery rate of 85% for the rotational speed of 1200 rpm. Although the hybrid design of separator has a better separation efficiency and higher purity, the passive one has no need for a time-consuming process of cell labeling, occupies less space on the disk, and does not impose additional costs and complexity.

Gene expression profiling as a potential predictor between normal and cancer samples in gastrointestinal carcinoma

Analysis and comparison of gene expression profile among molecules, correlated with essential and crucial biological processes, is of primary importance in cancer research, since it provides significant info regarding the resistance to chemo/radiotherapy, risk for relapse or prediction of metastasis etc. In this study, gene expression profile is used for discriminating efficiently colon cancer cell lines from normal cells and cancer cells in blood samples of colon cancer patients and categorizing different types of gastrointestinal cancer. In particular, blood samples were collected from normal donors as well as from colon cancer patients. Peripheral blood mononuclear cells were isolated and gene expression analysis was performed for more than fifty genes. The same assays were performed for commercial cancer cell lines representing different types of gastrointestinal cancer. In order to examine whether the comparison of gene expression profile can lead to a thorough discrimination between cancer and normal states as well as between different cancer types, we performed clustering analysis based on hierarchical, and k-means algorithms. The clustering analysis efficiently separated: a) colon cancer cell lines from colon patients' samples, b) normal from the colon cancer samples, c) gastric and pancreatic cancer from liver and colon types based. The exploitation of gene expression profile can be successfully used for the discrimination between normal vs cancer samples and/or for categorizing various types of cancer. This of course has important implications in cancer management since it enables the quick discrimination based on cells, isolated from bloodstream, needless of tissue examination or protocols requiring specialized equipment.

Abstract 5576: Isolation and detection of breast cancer cells in blood samples using antibody cocktails selected by large-scale genome-wide screening

Abstract Screening and prevention is critical to reducing breast cancer deaths, and mammography is not an ideal tool. Mammography has not reduced invasive breast cancer incidence, it has high numbers of false positives and it detects many non-invasive cancers that are not life threatening. Metastatic breast cancer is the second leading cause of cancer death for women. The development of a new blood test for breast cancer screening could offer significant improvements with the potential to be more accurate, less costly, less intimidating, not subject to age differences, and able to discriminate between cancers subtypes. Detection of breast cancer cells (BCC) in blood samples offers a valuable opportunity for screening. However, BCC in blood are rare and must be identified against a background of millions of other blood cells. Although many technologies have been developed for the isolation and detection of circulating tumor cells using EpCAM and cytokeratins, these tests do not provide levels of sensitivity and specificity required for a cancer screening tool. To address this critical unmet need, we developed a panel of cell surface markers whose expression patterns specifically distinguish breast cancer cells in blood samples. These genes were selected based on a discovery platform tailored to detect early stage breast cancers and include 20 novel putative cell surface molecules that are candidates for breast cancer cell detection and isolation. We prioritized these molecules by specificity of expression in tumor cells including: 1) Ratio >25:1 average breast tumor biopsy expression versus average normal blood expression, 2) High expression relative (>3 fold) in >20% of tumor biopsy samples, 3) High expression (>3 fold) in >40% of breast cancer cell lines, and 4) Expression significantly associated with 10-year outcomes (Kaplan Meier analysis, p<0.05). We combined the top 10 ranking genes in each of these categories, resulting in a list of 20 candidates that represent high priority targets. Antibodies for all 20 genes are commercially available. Expression in micro dissected biopsies was markedly higher than non-breast cancer blood for most. Also, most were expressed at lower levels than EpCAM in normal breast tissue and had background level expression in isolated normal blood cells. The innovation of this strategy is twofold. First, it uses a panel of molecules instead of an individual molecule to detect and isolate breast cancer cells and, second, it relies on molecules that are clinically relevant to breast cancers, rather than relying on generic epithelial cell markers. Current studies include the validation of antibody cocktails with specificity and sensitivity that outperforms the current EpCAM enrichment method and provides a proof of feasibility for screening of breast cancer. Citation Format: Lucas Delmonico, Edward C. Goodwin, Marcia Vasconcellos Fournier. Isolation and detection of breast cancer cells in blood samples using antibody cocktails selected by large-scale genome-wide screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5576.

Abstract 2760: Proof of principle studies for detection of circulating renal cancer cells from blood samples using diverse technologies

Abstract Background: During the past years, much progress has been made in detection and analysis of circulating tumor cells (CTCs) in several tumor entities, including prostate cancer, breast cancer or lung cancer. However, little is known about circulating tumor cells in patients suffering from clear cell renal cell carcinomas. The majority of technologies detecting CTCs is based on expression of epithelial markers on the surface of these cells, e.g. EpCAM. Additionally, biophysical approaches have been invented to detect CTCs based on size, invasive capacity or density. In order to be able to detect CTCs in patient samples, in vitro establishment of the most accurate isolation procedure followed by precise detection techniques has to be performed. Aim of our studies was to build a stable in vitro fundament of isolation and subsequent detection of CTCs in ccRCC patients. Methods: We made use of 4 different technologies, all of which have been approved for detection of CTCs in distinctive tumor entities. We used EpCAM based positive enrichment of CTCs, Ficoll densitiy centrifugation followed by CD45-positive cell depletion, rosette formation followed by CD45 positive cell depletion as well as size and deformability based enrichment technologies by using the Parsortix system. Furthermore, by using 4 phenotypically distinct ccRCC cell lines, we tried to detect markers unique for tumor cells in demarcation to blood cells. Results: By performing spiking experiments of renal cancer cells, we found the highest recovery rates by using the size based Parsortix system. Interestingly, the most established technique of EpCAM based isolation failed in three out of four cell lines to recover more than 40%. Expression of well-established markers for ccRCC, like carboanhydrase (CA)-9, could be detected in renal cancer cell lines. However, expression was also found in blood samples of healthy donors. Another marker used for immunohistochemical diagnosis of ccRCC, PAX8, showed weak to absent expression in established renal cancer cell lines. The highest specificity to detect renal cancer cells in blood samples was found when analyzing KRT8 or KRT 19 expression. Conclusion: Our results demonstrate that firstly, using the EpCAM based CTC enrichment, which is the basis of the CellSearch system, which up to now is the only methodology approved by the FDA, the majority of renal cancer cells will presumably not be detected in blood samples. This seems largely due to low or absent expression of EpCAM on renal cancer cells. The usage of the size based Parsortix system showed the highest recovery rates and should therefore be analyzed in more detail on samples of ccRCC patients. Secondly, an exclusive marker for defining a renal CTC is still missing. Some well-established ccRCC markers, like CA-9, failed to specifically detect renal cancer cells in blood samples, as they were either present also in healthy blood samples or absent in renal cancer cell lines. Citation Format: Yvonne Maertens, Verena Humberg, Julie Steinestel, Martin Boegemann, Andres J. Schrader, Christof Bernemann. Proof of principle studies for detection of circulating renal cancer cells from blood samples using diverse technologies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2760. doi:10.1158/1538-7445.AM2017-2760

Circulating tumor cell detection: A direct comparison between negative and unbiased enrichment in lung cancer.

Circulating tumor cells (CTCs), isolated as a 'liquid biopsy', may provide important diagnostic and prognostic information. Therefore, rapid, reliable and unbiased detection of CTCs are required for routine clinical analyses. It was demonstrated that negative enrichment, an epithelial marker-independent technique for isolating CTCs, exhibits a better efficiency in the detection of CTCs compared with positive enrichment techniques that only use specific anti-epithelial cell adhesion molecules. However, negative enrichment techniques incur significant cell loss during the isolation procedure, and as it is a method that uses only one type of antibody, it is inherently biased. The detection procedure and identification of cell types also relies on skilled and experienced technicians. In the present study, the detection sensitivity of using negative enrichment and a previously described unbiased detection method was compared. The results revealed that unbiased detection methods may efficiently detect >90% of cancer cells in blood samples containing CTCs. By contrast, only 40-60% of CTCs were detected by negative enrichment. Additionally, CTCs were identified in >65% of patients with stage I/II lung cancer. This simple yet efficient approach may achieve a high level of sensitivity. It demonstrates a potential for the large-scale clinical implementation of CTC-based diagnostic and prognostic strategies.

Nanoroughened adhesion-based capture of circulating tumor cells with heterogeneous expression and metastatic characteristics.

BackgroundCirculating tumor cells (CTCs) have shown prognostic relevance in many cancer types. However, the majority of current CTC capture methods rely on positive selection techniques that require a priori knowledge about the surface protein expression of disseminated CTCs, which are known to be a dynamic population.MethodsWe developed a microfluidic CTC capture chip that incorporated a nanoroughened glass substrate for capturing CTCs from blood samples. Our CTC capture chip utilized the differential adhesion preference of cancer cells to nanoroughened etched glass surfaces as compared to normal blood cells and thus did not depend on the physical size or surface protein expression of CTCs.ResultsThe microfluidic CTC capture chip was able to achieve a superior capture yield for both epithelial cell adhesion molecule positive (EpCAM+) and EpCAM- cancer cells in blood samples. Additionally, the microfluidic CTC chip captured CTCs undergoing transforming growth factor beta-induced epithelial-to-mesenchymal transition (TGF-β-induced EMT) with dynamically down-regulated EpCAM expression. In a mouse model of human breast cancer using EpCAM positive and negative cell lines, the number of CTCs captured correlated positively with the size of the primary tumor and was independent of their EpCAM expression. Furthermore, in a syngeneic mouse model of lung cancer using cell lines with differential metastasis capability, CTCs were captured from all mice with detectable primary tumors independent of the cell lines’ metastatic ability.ConclusionsThe microfluidic CTC capture chip using a novel nanoroughened glass substrate is broadly applicable to capturing heterogeneous CTC populations of clinical interest independent of their surface marker expression and metastatic propensity. We were able to capture CTCs from a non-metastatic lung cancer model, demonstrating the potential of the chip to collect the entirety of CTC populations including subgroups of distinct biological and phenotypical properties. Further exploration of the biological potential of metastatic and presumably non-metastatic CTCs captured using the microfluidic chip will yield insights into their relevant differences and their effects on tumor progression and cancer outcomes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2638-x) contains supplementary material, which is available to authorized users.

Detection of Breast Cancer Cells in Blood Samples by Immunostaining of the Thomsen–Friedenreich Antigen

Disseminated tumor cells are found in the bone marrow of patients with epithelial carcinoma and are correlated with a poor prognosis of the disease. Their detection is a technical challenge. This report describes a model system for the detection of cancer cells by co-immunostaining of Thomsen-Friedenreich and Her-2 antigens. Small numbers of cancer cells from different cancer cell lines were mixed with blood samples of healthy donors. Cytospins were prepared and double immunostaining against Thomsen-Friedenreich antigen and Her-2 was carried out by fluorochrome-coupled antibodies. Quantification of Thomsen-Friedenreich and/or Her-2-positive cells was performed with an epifluorescence microscope. On average, 83% of cancer cells were recovered by this method. Immunostaining is a useful method for the detection of cancer cells in blood samples. Results of this model system will be transferred to bone marrow patient samples to prove the benefits for detection of disseminated tumor cells.

278 Two Methods for the Detection of Breast Cancer Cells in Blood Samples

278 Two Methods for the Detection of Breast Cancer Cells in Blood Samples

P1-07-24: Quantitative and Immunohistochemical Detection of Breast Cancer Cells in Blood Samples.

Abstract Background Disseminated tumor cells (DTCs) in bone marrow and circulating tumor cells (CTCs) in blood are found in patients with epithelial carcinomas (breast cancer) and are often correlated with poor prognosis of the disease. In current models circulating tumor cells (CTCs) dissolved from the primary tumor are thought to be responsible for the occurrence of metastases. However, the detection of CTCs is still a technical challenge. In this study, two methods for tumor cell detection of patients’ samples are presented (Real-Time-PCR and immunohisto-chemical staining). Both are simple and known methods with a high sensitivity and a spread marker panel. Materials and Methods For the implementation of both methods, different breast cancer cell lines have been used (Cama-1, MCF-7; ZR-75-1). For Real-Time-PCR, blood samples of a healthy donors were spiked with different cell counts (0, 10, 100, 1000, 10.000 and 100.000) per ml blood sample. Total RNA was isolated from the samples, reversely transcribed into cDNA and used for TaqMan Real-Time-PCR reaction with probes against CK8, 18 and 19, while 18S was used as reference. Relative Quantification Curves were drawn by Microsoft™ Excel®. For immunohistochemical staining, cytospins were prepared from spiked blood samples, fixed with acetone, air dried and stained with antibodies against Her2- and Thomsen-Friedenreich-Antigen (CD176). In a second staining step fluorescently labelled secondary antibodies were applied. Nuclei were counterstained with DAPI, TF-Antigen was stained by Cy2 and Her2-Antigen by Cy3. The staining was controlled and documented by an epifluorescence microscope. Results The curve of Relative Quantification for MCF-7 and ZR-75-1 cells shows an increasing slope starting from 1000 cells. For the Cama-1 cell line this trend is already seen from 10–100 cells. In ZR-75-1 all three genes analysed reveal this trend, whereas in Cama-1 and MCF-7 cells a strong increase in Relative Quantification is especially seen for CK8 and 18. In the immunohistochemical staining, the cells were considered as tumour cells if they showed staining with the antibody-combinations used. Stained cells were counted and recovery rates were determined. For ZR-75-1, 17 of 30 cells which were spiked in the blood samples were recovered. For MCF-7, 18 cells were found in average, and for Cama-1 23 cells were located per slide. The recovery rates calculated from these numbers are 56,6% and 60,0% for ZR-75-1 and MCF-7, for Cama-1 the recovery rate reaches 76,6%. Conclusion It seems that Cama-1 cells are a better model than MCF-7 and ZR-75-1 for Real-Time PCR quantification of mamma carcinoma tumor cells in blood samples. MCF-7 and ZR-75-1 cells tend to react more likely immunologically with blood cells of the donor (agglutination between blood cells and cancer cells). The Cama-1 cell line shows also advantages in the detection of tumor cells using immunohistochemical staining. Therefore it will be necessary to test both methods on patient samples to proof their benefit. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-24.

Bioconjugated carbon nanotubes for targeting cancer biomarkers

This paper presents real-time detection of breast cancer cells through label-free protein biosensors based on antibody-conjugated carbon nanotube field effect transistors (CNTFETs). Both insulin-like growth factor 1 receptor (IGF1R) specific and nonspecific monoclonal antibodies (mAbs) reduced the electrical conductance of the CNTFET devices. Subsequent application of human BT474 and MCF7 breast cancer cells resulted in an increase in the conductance only for the IGF1R-specific mAb-CNTFET devices. Furthermore, the MCF7 breast cancer cells produced a greater increase in device conductance compared to BT474 breast cancer cells. This could be attributed to the larger number of IGF1 receptors in MCF7 breast cancer cells compared to BT474 cancer cells. Nonspecific mAb-CNTFETs did not yield any change in the conductance of the devices due to application of BT474 and MCF7 breast cancer cells. These results therefore imply that specific binding between IGF1 receptor and its specific mAb results in a dramatic change in electrical conductance of CNTFET devices. Consequently, mAb-CNTFETs are very promising biosensor candidates to detect circulating cancer cells in blood samples.

Automated wavelet denoising of photoacoustic signals for circulating melanoma cell detection and burn image reconstruction

Photoacoustic image reconstruction may involve hundreds of point measurements, each of which contributes unique information about the subsurface absorbing structures under study. For backprojection imaging, two or more point measurements of photoacoustic waves induced by irradiating a biological sample with laser light are used to produce an image of the acoustic source. Each of these measurements must undergo some signal processing, such as denoising or system deconvolution. In order to process the numerous signals, we have developed an automated wavelet algorithm for denoising signals. We appeal to the discrete wavelet transform for denoising photoacoustic signals generated in a dilute melanoma cell suspension and in thermally coagulated blood. We used 5, 9, 45 and 270 melanoma cells in the laser beam path as test concentrations. For the burn phantom, we used coagulated blood in 1.6 mm silicon tube submerged in Intralipid. Although these two targets were chosen as typical applications for photoacoustic detection and imaging, they are of independent interest. The denoising employs level-independent universal thresholding. In order to accommodate nonradix-2 signals, we considered a maximal overlap discrete wavelet transform (MODWT). For the lower melanoma cell concentrations, as the signal-to-noise ratio approached 1, denoising allowed better peak finding. For coagulated blood, the signals were denoised to yield a clean photoacoustic resulting in an improvement of 22% in the reconstructed image. The entire signal processing technique was automated so that minimal user intervention was needed to reconstruct the images. Such an algorithm may be used for image reconstruction and signal extraction for applications such as burn depth imaging, depth profiling of vascular lesions in skin and the detection of single cancer cells in blood samples.

Serial monitoring for circulating cancer cells in blood samples of stage 1–4 breast cancer patients

623 Serial monitoring for presence, number & characterization of circulating cancer cells (CCC) may provide valuable information that may be relevant to prognosis and treatment outcomes of breast cancer patients (BCP). We conducted a serial blood sampling study at the University of Maryland in BCP with stage 1–4 breast carcinoma. 15–20 ml of venous blood were collected before the start of systemic therapy and periodically thereafter & processed using negative selection method with double-gradient centrifugation & magnetic cell sorting to remove WBCs. Digital images of FITC-positive epithelial cells were acquired with a fluorescence microscope & counted. CCC from 41 patients (Pts) were also stained with Trastuzu-mAb-532 to quantify the HER-2/neu cell surface receptor expression relative to a fluorescence standard. 105 Pts were accrued & 415 blood samples tested (median number of samples/pt; 4 (1–8). During the 24 mos. monitoring period CCC were detected in 57 of 105 pts (54%). The Table below shows that presence of >10 CCC/sample is associated with decreased survival and increased probability of having metastatic disease.(Exact chi-square test for presence vs. absence of metastatses in A, B, C, D groups, P < 0.0001; Fisher’s exact test to compare individual groups: for B vs C+ D, P < 0.001; B vs C, P=0.001). HER-2/neu expression was assessed in CCC of 25 pts (minimum of 4 CCC per sample) as compared with strongly HER-2/neu positive control cell line SKBR-3. 10 Pts were positive & 15 negative for HER-2/neu over-expression in CCC. CCC data & primary tumor data concurred in 6 of 7 Her-2/neu primary tumor tissue positive Pts & in 12 of 13 Her-2/neu primary tissue negative Pts. For 5 Pts tissue data was not available. Conclusions: Increasing CCC numbers/sample appear to correlate with adverse outcome of BCP. Our CCC Test may provide valuable information about prognosis of stage 1–4 BCP. HER-2/neu expression could be quantified in individual CCC & concurred with primary tumor data in 90% of Pts. Supported by NCI Grant CA081903 [Table: see text] [Table: see text]

Single-Wall Carbon Nanotubes with Adsorbed Antibodies Detect Live Breast Cancer Cells

Monoclonal antibodies (mAb) specific to cell surface antigens overexpressed on cancer cells adsorbed to single-wall carbon nanotube (SWCNT) devices can bind to their antigens in a drop of buffer, resulting in a slight drop in conductance. Here we report detection of live breast cancer cells with a mAb-SWCNT device. We adsorbed mAb specific to insulin-like growth factor 1 receptor (IGF1R) onto interconnected SWCNT networks placed between lithographically patterned electrodes. Application of human BT474 breast cancer cells increased the conductance of the IGF1R-specific mAb-SWCNT devices by 3.0±0.1-fold, relative to nanotube devices with non specific mAb. Human MCF7 breast cancer cells, with greater IGF1R expression, increased the conductance by 8.0±0.2-fold, but R-cells lacking IGF1R did not. Receptor-specific mAb acted as specific nanoswitches that completed a circuit between the SWCNT and the cell surface receptors, elevating the device conductance. Such devices might detect circulating breast cancer cells in blood samples.