This paper presents a reproducible method with new findings on the presence of epithelial cells in normal human and mouse blood and bone marrow using flow cytometry and immunofluorescence microscopy. Krt1-14;mTmG transgenic mice were used as an in vivo method to confirm these findings.
Epithelial cells have been identified in the blood and bone marrow of patients with cancer and other diseases. However, the presence of normal epithelial cells in the blood and bone marrow of healthy individuals has yet to be identified in a consistent way. Presented here is a reproducible method for isolating epithelial cells from healthy human and murine blood and bone marrow (BM) using flow cytometry and immunofluorescence (IF) microscopy. Epithelial cells in healthy individuals were first identified and isolated via flow cytometry using epithelial cell adhesion molecule (EpCAM). These EpCAM+ cells were confirmed to express keratin using immunofluorescence microscopy in Krt1-14;mTmG transgenic mice. Human blood samples had 0.18% ± 0.0004 EpCAM+ cells (SEM; n=7 biological replicates, 4 experimental replicates). In human BM, 3.53% ± 0.006 (SEM; n=3 biological replicates, 4 experimental replicates) of mononuclear cells were EpCAM+. In mouse blood, EpCAM+ cells constituted 0.45% ± 0.0006 (SEM; n=2 biological replicates, 4 experimental replicates), and in mouse BM, 5.17% ± 0.001 (SEM; n=3 biological replicates, 4 experimental replicates) were EpCAM+. In mice, all the EpCAM+ cells were immunoreactive to pan-cytokeratin, as determined by IF microscopy. Results were confirmed using Krt1-14;mTmG transgenic mice, with low (8.6 native GFP+ cells per 106 cells analyzed; 0.085% of viable cells), but significant numbers (p < 0.0005) of GFP+ cells present in normal murine BM, that were not the result of randomness compared with multiple negative controls. Further, EpCAM+ cells in mouse blood were more heterogeneous than CD45+ cells (0.58% in BM; 0.13% in blood). These observations conclude that cells expressing cytokeratin proteins are reproducibly detectable among mononuclear cells from human and murine blood and BM. We demonstrate a method of tissue harvesting, flow cytometry, and immunostaining that can be used to identify and determine the function of these pan-cytokeratin epithelial cells in healthy individuals.
Epithelial cells are found in the physical barriers between our bodies and the environment and are able to recognize and respond to changes in their microenvironment1. They have a proliferating stem cell niche which provides a way to turn over new tissue and repair damage2. Our lab studies stem cells in the hair follicles of the skin; skin is a good model for studying epithelial tissue and stem cell proliferation because it is easily visible and there is a constant turnover of cells. Epithelial cancers are the most common form of cancer, possibly due to epithelial tissues, such as the skin, being the first line of defense against environmental carcinogens, leading to high turnover rates and the proliferation of epithelial cells3. Most of the skin epidermis, the top protective layer, is composed of keratinocytes, which express different types of keratins to provide support and structure4. Patients with epithelial cancers often have epithelial cells present in their blood and bone marrow that also express keratins. Liquid biopsies are a noninvasive way to detect and monitor these epithelial cells in different bodily fluids5. Circulating epithelial cells, also called circulating tumor cells (CTCs), are found in peripheral blood and can be biomarkers for cancer prognosis, as well as guide individualized therapy treatments. CTCs can also indicate disease progression, treatment efficacy, and overall patient survival5,6.
Epithelial cell adhesion molecule (EpCAM) is a clinically used marker for CTCs and can identify tumors of epithelial origin in cancer patients. EpCAM plays a role in cell adhesion, migration, signaling, proliferation, and differentiation6. For a circulating epithelial cell to be classified as a CTC, it must be positive for cytokeratins 8, 18, and 19, and negative for CD45, a common leukocyte marker6. CTCs are usually identified by first depleting CD45 with magnetic microbeads, followed by testing for EpCAM and cytokeratin 19 using immunofluorescence microscopy7. The main limitation in the detection of CTCs is their rarity; they make up less than 0.01% of all cells in the blood, and very few survive in circulation to reach distant organs8,9,10. Consideration must be taken in designing experiments and techniques to isolate and identify these cells due to their rare nature. Currently, there is only one Food and Drug Administration (FDA)-approved automated single-cell sorter used for identifying CTCs, and it uses EpCAM as its biomarker. Other methods include magnetic bead separation and flow cytometry, or a combination of these methods. There is a need for new techniques that have higher sensitivity and specificity for the detection of rare CTCs11.
Flow cytometry is a preferred method for the detection of rare cell populations in the blood, bone marrow, and other tissue samples. These rare cells can include stem cells, circulating endothelial cells, CTCs, and residual disease cells. Flow cytometry enables quantitative measurements of each cell type and sorts these cells for further testing7,9. Incremental counts were performed to ensure an accurate assessment of these rare cells. The ability to use gating to exclude cells from further analysis is a way to increase specificity when analyzing cells. The limitations of flow cytometry are the time required for the analysis of large samples and the lack of visual confirmation for cell identity. To overcome this, immunofluorescence microscopy was performed on the sorted cells to confirm their identities.
Previously our lab has shown that in mice the bone marrow cells are recruited and contribute to skin tumors12. These bone marrow-derived cells are positive for pan-cytokeratin and epidermal cytokeratin. To further elucidate the role of epithelial stem cells, bone marrow-derived cells, and cytokeratin-expressing cells in tumor progression, EpCAM+ cytokeratin-positive cells in normal murine and human blood and bone marrow samples were looked for. As with most experiments, this method was developed through multiple iterations. Previously, transgenic mice were used along with incremental counts to look for K14GFP-expressing cells in the bone marrow12. As more cells were counted, a representative population of rare cells was able to be identified, as shown in Figure 1 in the representative results section. The rationale for investigating epithelial cells in normal blood and bone marrow was based on early literature on CTCs, where normal healthy donors had background levels of EpCAM+ cells13. As mentioned previously, EpCAM characterization often begins with the depletion of CD45. This step was omitted because some hematopoietic cells have EpCAM and cytokeratin expression for unknown reasons that need to be investigated further. Therefore, cells were sorted based on the presence or absence of EpCAM, irrespective of cell lineage, and then immunostained for cytokeratin. The protocol below and the workflow shown in Figure 2 describe a technique that uses flow cytometry with compensation and controls, statistical methods, and immunofluorescence imaging to isolate and identify these rare populations of epithelial cells.
There is some evidence in the literature of the presence of epithelial cells in the bone marrow. Previously, papers have typically investigated the role of epithelial cells within the context of disease and injury, such as in the liver, lung, gastrointestinal (GI) tract, thymus, and skin14,15,16,17. However, not much is known about the presence of these epithelial cells in the bone marrow of healthy individuals. This paper seeks to establish a reproducible method, with the aim of identifying and isolating epithelial cells from normal blood and bone marrow. This method will drive the field forward to identify why epithelial cells are present and what their role is in the blood and bone marrow in the absence of disease; perhaps these cells are part of normal tissue maintenance or activated at times of injury. The bone marrow is a repository of stem cells; however, it is unclear what the lineage of these epithelial cells may be. A recent paper discusses bone marrow-derived epithelial cells in the thymus, that first expresses EpCAM and the hematopoietic marker CD45, and then loses its CD45 expression over time after injury15. Research from our lab has also confirmed the presence of CD45+ EpCAM+ cells within healthy blood and bone marrow in the absence of injury12. However, the role of these cells is yet to be determined.
There was a need for a reproducible method to examine epithelial cells within the healthy bone marrow. The method described will help in characterizing these cells further in their normal state. There are important steps in this method to maintain reproducibility. One of the most critical steps in this protocol is maintaining a sterile environment in the hood while harvesting the bone marrow from mice. If harvesting from multiple mice, cross-contamination between samples is avoided by using new needles and syringes for each mouse. This also ensures that the sample is clean and free of any contaminants that might affect the results. In addition, the number of prepared syringes and labeled conical tubes for each additional mouse should be increased. Another important step involves flushing the bones until a clean white color is evident to ensure that most of the bone marrow cells have been removed; the chances of detecting rare cells increases in a purer sample. A modification was made to optimize the red blood cell lysis protocol; several lysis buffers were tested to find the right one that gave consistently high viability, as almost half the cells were being lost during this step. Different reagents and incubation periods may need to be optimized for improved results in other settings.
The most significant limitation of this protocol is using flow cytometry for finding rare cell populations. As discussed previously, the addition of controls and incremental counts helps increase the specificity and accuracy of the analysis. Another limitation is that appropriate markers for the population of interest must be identified in advance. Thus, one needs to know about key markers, antibodies for flow cytometry, and antibodies compatible with the target species.
These methods are an improvement over existing methods as they allow for single cell analysis at a fraction of the cost of the existing automatic CTC isolator and single cell RNA sequencing. Additionally, flow cytometry is more readily available. FACS maintained higher viability for the cells compared with prior reported results using magnetic microbead separation. Lastly, these techniques allow for the separation of cells for downstream analyses, such as bulk RNA sequencing, scRNA sequencing, or cell culture.
The authors have nothing to disclose.
Josh Monts, Core Facility Flow Cytometer Technician, The Hormel Institute
Todd Schuster, Core Facility Manager, The Hormel Institute
Derek Gordon, Statistician, Rutgers University
We would like to thank Karen Klein from Clarus Editorial Services, Santa Fe, NM for her editorial assistance.
This work was supported in part by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number R21 AR075281, and a Grant-in-Aid of Research, Artistry and Scholarship, Office of the Vice President for Research, University of Minnesota (Proposal #324240). We gratefully acknowledge support from The Hormel Institute.
40 μm Cell Strainer | Falcon | 352340 | Used to filter out any large clumps of cells from bone marrow |
5 mL Polystyrene Round-bottom tube | Falcon | 352054 | Tubes used for flow cytometry |
500 mL jar with lid | Nalgene | 11-823-32 | Used to wash the mouse |
190 proof Ethanol | Decon laboratories Inc | 2801 | Diluted to 70% with dH2O |
Alexa Fluor 488 Goat anti-rabbit IgG (H+L) | Invitrogen | A11008 | Dilution: 1:1000 |
Brightline Hemacytometer | Hausser Scientific | 02-671-10 | Used to count alive cells |
Bovine Serum Albumin | Jackson ImmunoResearch | 001-000-162 | Component of antibody diluent |
Characterized Fetal Bovine Serum (FBS) | Hyclone | SH30071.03 | Component of staining buffer and harvesting solution |
Curity gauze sponges | Covidien | 2187 | Used to keep a clean work area when harvesting limbs and bone marrow |
Curved Forceps | Miltex | 18-784 | Used during harvest |
Cytokeratin, Wide Spectrum Screening | Dako | Z0622 | Dilution 1:750 *Now discontinued |
Dulbecco's Phosphate Buffered Saline (DPBS 1x) | Gibco | 14190-144 500mL | Used to stop the red blood cell lysis reaction |
Ethylenediaminetetraacetic acid | Sigma Aldrich | E9884-100G | Used at 0.5 M |
Gentamicin sulfate | Lonza | 17-518Z | Component of harvesting solution |
Hank's Balanced Salt Solution (HBSS 1x) | Gibco | 14175-095 500mL | Component of harvesting solution and staining buffer solution |
Luer-Lok tip 10 mL syringe | Becton, Dickinson and Co | 309604 | Used with 26G needle to flush bones, used with 20G needle to break up clumps |
Magic touch 2 ice bucket | BelArt | M16807-2001 | Used to store the specimens on ice |
Nonfat dry milk | Apex | 20-241 | Component of Antibody Diluent |
Normal horse serum | Vector | ZE0122 | Component of Antibody Diluent |
PE anti-human CD 326 (EpCAM) | Biolegend | 324206 | Dilution: 5 µl in 100 µl per 1×106 cells |
PE anti-mouse CD 326 (EpCAM) | Biolegend | 118205 | Dilution: 3 µl in 100 µl per 1×106 cells |
PE mouse IgG2b, κ isotype control | Biolegend | 400313 | Dilution: 1 µl in 100 µl per 1×106 cells |
PE rat anti-human CD49f | BD Biosciences | 555763 | Dilution: 20 µl in 100 µl per 1×106 cells |
PE rat IgG2am, κ Isotype control | Biolegend | 400508 | Dilution: 1 µl in 100 µl per 1×106 cells |
Polypropylene Conical Centrifuge Tubes 50 mL | Basix | 14-955-240 | Used in the centrifuge |
Povidone-Iodine Scrub | Aplicare | 82-227 | Antiseptic used to sterilize the mice |
PrecisionGlide Needle 20G x 1 1/2 | Becton, Dickinson and Co | 305176 | 20G needle used to break up bone marrow clumps |
PrecisionGlide Needle 26G x 1/2 | Becton, Dickinson and Co | 305111 | 26G needle used to flush bone marrow from bones |
PTFE Printed Slides | Electron Microscopy Services | 63422-06 | 8 well slides cells sorted onto for immunofluorescence |
RBC Lysis buffer 10X | Invitrogen | 00-4300-54 | Dilution 1:10 using sterile deionized water |
Scissors | Roboz | RS-6762 | Used during harvest |
Stainless steel surgical blade #4 | Bard-Parker | 371222 | Used during harvest |
Sterile tray | Polar ware | 10F | Used during harvest |
StretchEase Powder-free nitrile examination gloves | Denville Scientific | G4161 | Used during harvest |
Surgical Scalpel handle #4 | Fischer | 12-000-164 | Used with surgical blade |
TBS 20x | Thermo | Component of TBST, used at 1X | |
Trypan Blue Stain (0.4%) | Gibco | 15250-061 | Used to count dead cells. Filtered with .45 µm |
Tween 20 | Sigma Aldrich | P1379-500mL | Component of TBST |
Tweezers | Miltex | 6-8 | Used during harvest |
Vectashield Vibrance Antifade Mounting Medium with DAPI | Vector | H-1800 | Nuclear stain for immunofluorescence |