Detecting cell-in-cell structures in human tumor samples by E-cadherin/CD68/CD45 triple staining

Abstract

Although Cell-in-cell structures (CICs) had been documented in human tumors for decades, it is unclear what types of CICs were formed largely due to low resolution of traditional way such as H&E staining. In this work, we employed immunofluorescent method to stain a panel of human tumor samples simultaneously with antibodies against E-cadherin for Epithelium, CD68 for Macrophage and CD45 for Leukocytes, which we termed as "EML method" based on the cells detected. Detail analysis revealed four types of CICs, with tumor cells or macrophage engulfing tumor cells or leukocytes respectively. Interestingly, tumor cells seem to be dominant over macrophage (93% vs 7%) as the engulfer cells in all CICs detected, whereas the overall amount of internalized tumor cells is comparable to that of internalized CD45+ leukocytes (57% vs 43%). The CICs profiles vary from tumor to tumor, which may indicate different malignant stages and/or inflammatory conditions. Given the potential impacts different types of CICs might have on tumor growth, we therefore recommend EML analysis of tumor samples to clarify the correlation of CICs subtypes with clinical prognosis in future researches.

Keywords: EML method; cell cannibalism; cell-in-cell structures; epithelium; macrophage.

Figure 1. E-cadherin/CD68/CD45 triple staining of stomach TMA

A. Images of human stomach TMA stained with antibodies for E-cadherin, CD68 and CD45. Nuclei were counterstained with DAPI. Left panel shows a stitched image of whole TMA with multiple fluorescent channels merged (a) and schematic illustration of tumor stage of each core (b); Middle panel shows representative core image of the TMA circled in (a), with the upper one (c) showing the raw scanned image and lower one (d) showing spectral-processed image of (c). The right panels (e-h) display images of four unmixed single fluorescent channels as indicated. Scale bar: 150 μm. B. Left image shows cropped region of core image (A-d), right images show four unmixed single fluorescent channels as indicated. Scale bar: 20 μm.

Figure 2. E-cadherin/CD68/CD45 triple staining of multiple TMAs

Multiple human tumor TMAs were stained with antibodies for E-cadherin, CD68 and CD45. Nuclei were counterstained with DAPI. Left two panels show the schematic illustrations of tumor stage of each core (stage pattern) and stitched images of whole TMA (scanned TMA), respectively. Right five panels show unmixed images of single fluorescent channels and merged composite images for the cores circled in scanned TMA panel. TMAs from tumors of liver, lung, colon, breast, pancreas and prostate were stained and displayed.

Figure 3. Profile of CICs detected in various tumor samples

A. Graph shows number of cores where CICs were detected in each tumor TMA. B. Quantification of total CICs detected in each tumor TMA. C. Average CICs number in each CICs-positive core for each tumor TMA. Sto Ca: stomach carcinoma; Liv Ca: liver carcinoma; Lun Ca: lung carcinoma; Col Ca: colon carcinoma; Bre Ca: breast carcinoma; Pan Ca: pancreas carcinoma; Pro Ca: prostate carcinoma.

Figure 4. Four types of CICs detected in human tumors

Representative images for each CICs subtype: CD45⁻/CD68⁻ A. CD45⁺/CD68⁻ B. CD45⁻/CD68⁺ C. CD45⁺/CD68⁺ D. White arrows indicate inner cells or their nuclei, yellow arrows indicate outer cells or their nuclei. Scale bar: 20 μm.

Figure 5. Macrophages positive in CD163 participate in CICs formation in human tumors

Representative image is displayed in merged or single pseudo-color channel. Graph shows quantification of expression of CD163 (n = 66) or NOS2 (n = 98) in the outer/host cells of CICs identified. Scale bar: 15 μm.

Figure 6. Analysis of CICs subtypes in human tumors

A. Pie graph shows the distribution of four CICs subtypes in all CICs indentified from various tumor TMAs. B. The distribution of four CICs subtypes in CICs identified in individual tumor TMA. C. The distribution of CD68⁺ CICs and CD68⁻ CICs identified in individual tumor TMA. D. The distribution of CD45⁺ CICs and CD45⁻ CICs identified in individual tumor TMA. Sto Ca: stomach carcinoma; Liv Ca: liver carcinoma; Lun Ca: lung carcinoma; Col Ca: colon carcinoma; Bre Ca: breast carcinoma; Pan Ca: pancreas carcinoma; Pro Ca: prostate carcinoma. n = 295.