DOI: 10.5281/zenodo.44884
title: Fluorescent Confocal Laser Scanning Microscopy Of White Blood Cells, Cancer Cell Line Mcf7, And Mixtures Of These Cells: A Model System For Circulating Tumor Cell Biomarker Evaluation V.1

description: This is a confocal laser scanning microscopy data set of white blood cells (leukocytes), the cancer cell line MCF7, and mixtures of these cells acquired on a Zeiss LSM 780 microscope in the University of Colorado Anschutz Medical Campus Advanced Light Microscopy Core. Cells are fluorescently labeled for DNA with DAPI (Sigma D9542), lipids with Bodipy 495/503 (Thermo Fisher D3922), the filament protein cytokeratin (CK) with pan-cytokertain-alexa555 antibodies (Cell Signaling Technologies 3478S) and the surface membrane antigen CD45 with CD45-alexa647 antibodies (Biolegend 304020). Bodipy was excited with a continuous wave (CW) 488 nm laser, alexa555 was excited with CW 561 nm laser, and alexa647 was excited with a CW 633 nm laser. The acquiring instrument does not have a CW 405 nm source so DAPI was excited by two photon process using a Coherent Cameleon ultrafast pulsed laser tuned to 765 nm. The objective used was a Zeiss Plan-Apochromat 20x, 0.8 NA, air.

The data consists of 4 channel 8x8 mosaic z-stacks. The Zeiss software performed stitching of the mosaics. These stitched data images are included and marked with _Stitched at the end. Those interested in performing the stitching themselves can do this with the raw data files (without the _Stitched). The jpeg images are processed from the stitched LSM images. The LSM files contain additional meta data on the experiment including power levels and acquisition settings.

The _Stiched .lsm files will load in ImageJ (tested with V.1.49) as 4 channel 3 stack images.

This data is a model system for evaluating the DNA/Lipids/CK/CD45 biomarker panel to identify circulating tumor cells (CTCs). The D- population of the model is the WBCs and the D+ population is the MCF7 cancer cell line. The amount of separation the biomarker panel plus analysis algorithm can produce between these populations (D+/D-) is an estimate the sensitivity and specificity of the biomarker panel plus algorithm to CTCs.

Experiments generating the data were performed over the course of 15 days. Peripheral blood samples were collected from the Gynecological Tissue and Fluid Bank (COMIRB 07-0935 / COMIRB 05-1081) from consenting patients undergoing surgery at the University of Colorado Hospital. Blood samples were used the same day they were collected. Blood samples were collected from 3 patients with benign conditions, labeled WBBN#, and 3 patients with ovarian cancer, labeled WBCA#. We do not expect there to be any difference in the isolated white blood cells samples prepared from the cancer and benign patients. Samples were stored at room temperature until white blood cells were isolated. Mixed samples were prepared by passaging a MCF7 flask and mixing it with isolated white blood cells before fixation. A schedule showing the time duration between collection, processing and imaging is included as “experimental schedule.gif”.

The MCF7 cancer cell line was a kind gift from Dr. Heide Ford. Genomic DNA was isolated from the MCF7 cell line after the experiment and sent for cell line authentication. The gDNA was a match to MCF7. The authentication report and data are included in this submission.

CD45 antibodies were exhausted on day 7. New antibody was purchased and received on day 8. The day 7 images only has labels for DAPI and Bodipy. The samples prepared with the old antibodies on days 4 and 7 were relabeled and imaged with the new antibodies on days 14 and 15. This labeling was also done to confirm the pan-CK antibodies remained good since they are dim in the MCF7 cells imaged on days 12 and 13. The pan-CK on days 14 and 15 looks the same as it did on days 5 and 7 confirming the antibodies are good.

Four of the filters containing cells were not sufficiently flat to be acquired with a 3 slice z-stack so a 5 slice z-stack was used. These files have been zipped to compress them under the 2 GB limit permitted by zenodo.org

Further information on how these samples were prepared, processed, and analyzed can be found in our associated 2016 SPIE Photonics West BIOS conference proceeding titled, “Quantitative image cytometry measurements of lipids, DNA, CD45 and cytokeratin for circulating tumor cell identification in a model system”, http://dx.doi.org/10.1117/12.2222317.

This work was supported by funding provided to the University of Colorado Cancer Center by the American Cancer Society and awarded as Institutional Research Grant Number 57-001-53, by funding provided by the Defense Advanced Research Projects Agency under grant number N66001-10-4035, and by funding provided by NIH/NCATS Colorado CTSI Grant Number TL1 TR001081. The University of Colorado Anschutz Medical Campus Advanced Light Microscopy Core is also supported in part by NIH/NCATS Colorado CTSI Grant Number UL1 TR001082. The funders had no role in the study design, data collection, analysis, or decision to publish.