An image-based flow cytometric approach to the assessment of the nucleus-to-cytoplasm ratio

Joseph A Sebastian; Michael J Moore; Elizabeth S L Berndl; Michael C Kolios

doi:10.1371/journal.pone.0253439

. 2021 Jun 24;16(6):e0253439. doi: 10.1371/journal.pone.0253439

An image-based flow cytometric approach to the assessment of the nucleus-to-cytoplasm ratio

Joseph A Sebastian ^1,^2,^3,^*, Michael J Moore ^1,^2,³, Elizabeth S L Berndl ^1,^2,³, Michael C Kolios ^1,^2,^3,^*

Editor: Vincenzo L’Imperio⁴

PMCID: PMC8224973 PMID: 34166419

Abstract

The nucleus-to-cytoplasm ratio (N:C) can be used as one metric in histology for grading certain types of tumor malignancy. Current N:C assessment techniques are time-consuming and low throughput. Thus, in high-throughput clinical contexts, there is a need for a technique that can assess cell malignancy rapidly. In this study, we assess the N:C ratio of four different malignant cell lines (OCI-AML-5—blood cancer, CAKI-2—kidney cancer, HT-29—colon cancer, SK-BR-3—breast cancer) and a non-malignant cell line (MCF-10A –breast epithelium) using an imaging flow cytometer (IFC). Cells were stained with the DRAQ-5 nuclear dye to stain the cell nucleus. An Amnis ImageStreamX® IFC acquired brightfield/fluorescence images of cells and their nuclei, respectively. Masking and gating techniques were used to obtain the cell and nucleus diameters for 5284 OCI-AML-5 cells, 1096 CAKI-2 cells, 6302 HT-29 cells, 3159 SK-BR-3 cells, and 1109 MCF-10A cells. The N:C ratio was calculated as the ratio of the nucleus diameter to the total cell diameter. The average cell and nucleus diameters from IFC were 12.3 ± 1.2 μm and 9.0 ± 1.1 μm for OCI-AML5 cells, 24.5 ± 2.6 μm and 15.6 ± 2.1 μm for CAKI-2 cells, 16.2 ± 1.8 μm and 11.2 ± 1.3 μm for HT-29 cells, 18.0 ± 3.7 μm and 12.5 ± 2.1 μm for SK-BR-3 cells, and 19.4 ± 2.2 μm and 10.1 ± 1.8 μm for MCF-10A cells. Here we show a general N:C ratio of ~0.6–0.7 across varying malignant cell lines and a N:C ratio of ~0.5 for a non-malignant cell line. This study demonstrates the use of IFC to assess the N:C ratio of cancerous and non-cancerous cells, and the promise of its use in clinically relevant high-throughput detection scenarios to supplement current workflows used for cancer cell grading.

Introduction

There is a need in cancer diagnostics for techniques that overcome the drawbacks of current conventional cancer cell assessment methods. Currently, histological assessment is the gold standard of assessing cell and tissue malignancy [1] but lacks speed, high-throughput, and can be prone to differing interpretation from pathologists. In addition, histology is an inefficient technique in clinically relevant contexts that require high-throughput cellular analysis such as diagnostics of hematological diseases [2], diagnosis of minimal residual disease [3] and circulating tumor cell detection [4]. Thus, the utilization of high throughput techniques to assess cell malignancy may improve cancer diagnostics by providing cellular information of these rare phenotypes.

Clinicians have identified an enlarged nucleus as a prevalent characteristic of certain types of malignant cells [5–7]. The enlarged nucleus of these malignant cells led to the development of the nucleus-to-cytoplasmic (N:C) ratio, defined as the ratio of the cross-sectional area of the nucleus divided by that of the cytoplasm [8]. Although histology and cytology are the gold standards of the N:C ratio assessment method, many studies have reported the interobserver variability that exists in during visual quantitation [9–11]. In practice, cytology, where slides of biological specimen are fixed to a glass slides and examined, is another method used to assess the N:C ratio. In addition, although this metric is used in many tissue types (e.g., urothelial carcinoma), in others (e.g., melanoma), lower N:C ratios [12] despite malignancy and higher N:C ratios in normal cells (e.g., lymphocytes) prevent adoption of the N:C ratio as a grading method. Nevertheless, new techniques with less subjectivity have been developed to assess and quantify the N:C ratio of cancer cells (e.g., computer vision, two-photon microscopy, immunohistochemistry analysis techniques) [13–18]. Since these techniques rely on histological sectioning to assess the N:C ratio, they lack translatability to high-throughput clinical contexts where a liquid biopsy would be used to assess malignancy. A high-throughput technique that can assess cell malignancy using cells in suspension would be ideal. Imaging flow cytometry (IFC) can provide such a cytometric assessment method due to the high-throughput collection of images of single cells. IFC is a hybrid technology that combines conventional flow cytometry (FC) with high-throughput microscopy to generate high-resolution images of single cells in suspension within minutes [19, 20]. IFC combines the advantages of using FC with the ability to visually identify single cells. IFC has been used in many applications, including radiation biodosimetry, analysis of autophagy, and quantification of cellular heterogeneity [21–24]. In this work we demonstrate how IFC can be used to assess the N:C ratio of several malignant cell lines and a single non-malignant cell line.

Previously, our group has used IFC to compare cell size measurements with measurements done by photoacoustic microscopy and photoacoustic flow cytometry which were used for the N:C analysis of cultured breast and prostate cancer cells [25–27]. In this work, we determine the N:C ratio of four different malignant cell lines each originating from different tissues and the N:C ratio of a single non-malignant cell line. Here, acute myeloid leukemia (OCI-AML-5, blood cancer), CAKI-2 (kidney cancer), HT-29 (colon cancer), SK-BR-3 (breast cancer) cells, and MCF-10A (breast epithelial) cells were used for the measurements. Across cell lines, we observe varying cell and nuclear sizes but a common N:C ratio of ~0.6–0.7, consistent with international standards of diagnosis of urothelial carcinoma [7] and a smaller N:C ratio of 0.53 in the non-malignant cell type. This work demonstrates the diagnostic potential of IFC as an assessment technique of the N:C ratio and the promise of its use in clinically relevant high-throughput detection scenarios.

Methods

Cell preparation

In this work (1) SK-BR-3 (ATCC, Virginia, USA, HTB-30), HT-29 (ATCC, Virginia, USA), and CAKI-2 cells were thawed and cultured for two weeks in McCoy’s 5A (modified) Media (Wisent Inc., Quebec, Canada, 317-010-CL) supplemented with 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin, (2) OCI-AML-5 cells (DSMZ, Braunschweig, Germany, ACC247) were thawed and cultured for two weeks in Alpha modified Eagle medium (Wisent Inc., Quebec, Canada, 310-010-CL) containing 10% FBS and 1% penicillin-streptomycin (Wisent Inc., Quebec, Canada, 450-201-EL) by volume, and (3) MCF-10A cells MCF-10A cells (Addex Bio, California, USA, C0006015) were thawed and cultured for three weeks in 1:1 Dulbecco’s Modified Essential Media and F12 media (ThermoFisher, Massachusetts, USA, 11330032), supplemented with 5% Horse serum (Sigma-Aldrich, Ontario, Canada, H1270), 20 ng/mL EGF (Peprotech, New Jersey, USA, AF-100-15), 0.5 ug/mL Hydrocortisone (Sigma-Aldrich, Ontario, Canada, H0888), 100 ng/mL Cholera Toxin (List Biological Laboratories, California, USA, 100B), 10ug/mL Insulin (Sigma-Aldrich, Ontario, Canada, I1882), and 1% penicillin-streptomycin (Wisent Inc., Quebec, Canada, 450-201-EL). Once confluent, all adherent cells were trypsinized and resuspended in phosphate-buffered saline (PBS). The cells were stained using a 1:200 solution of DRAQ-5 (Thermo Fisher, Mississauga, Canada), a fluorescent nuclear dye and resuspended in a volume of 50 μl PBS and 1% FBS in a 1.5 ml low retention microfuge tube (MilliporeSigma, Oakville, Canada).

Image flow cytometer operating parameters and post-processing

The imaging parameters and post-processing for this work have been previously described [26]. Briefly, An Amnis ImageStreamX® MarkII IFC (MilliporeSigma, Seattle, USA) equipped with a 5-laser 12-channel system at 60x magnification, following ASSIST calibration (MilliporeSigma, Seattle, USA) was used for image acquisition. In this study, channels 1 to 11 were used for acquisition along with a 642-nm laser (150 mW); however, analysis on only channel 1 (430 to 480 nm), 7 (430–505 nm), and 11 (660–740 nm) were completed (Ch 1/7 –malignant cells, Ch1/11 –non-malignant cells). Since both Channel 7 and 11 are used for fluorescent imaging in IFC, there is no difference between usage of either channel. A bright-field area lower limit of 50 μm² was used to eliminate debris and speed beads during acquisition.

Cell image analysis was carried out using the Amnis IDEAS® software platform (version 6.2). An overview of the analysis workflow is shown in Fig 1A. The nucleus diameter and cell diameter were determined using a custom workflow in IDEAS, which is illustrated in Fig 1 and adapted from our previous work [26]. Briefly, from Fig 1.a.I, the gradient root-mean squared feature was applied to the acquired OCI-AML-5, CAKI-2, HT-29, SK-BR-3, and MCF-10A cell images, and the corresponding values were plotted on a normalized relative frequency distribution to remove unfocused cell images. Fig 1.a.II depicts the area and aspect ratio features combined to remove images containing multiple cells. In our workflow, we included cell images with an aspect ratio between approximately 0.55 and 1 to avoid cell fragments and other debris. Fig 1.a.III shows the raw centroid X feature plotted against a normalized relative frequency distribution to remove clipped cell images. The raw centroid X feature quantifies the central location of the acquired images. Lastly, Fig 1.a.IV depicts a positive gate for DRAQ-5-positive cells that was obtained using fluorescence intensity and area features. Through gating for solely DRAQ-5-positive cells in plot IV, we exclude cell images containing calibration beads, which are required for alignment of the sample stream during imaging. The rationale for this gate is based on the visual clustering of cells we see in the scatter plot based on area and high pixel intensity. Masks used for the image analysis process following the same protocol as our previously published work to accurately measure cell diameter and can be seen in Fig 2B–2D [25–27]. Here, the eroded masks were used to determine the cell diameter where IDEAS provided the diameter of a circle that has the same area as the eroded masks [28]. DRAQ-5 leakage out of the nucleus was addressed in our workflow for the non-malignant cells by adding a plot that used gradient root-mean-squared feature on the fluorescent images to isolate cells that had localization of the DRAQ-5 dye in their nucleus (high gradient RMS value). This plot is provided in Fig 1B. We applied this extra gate on all cell lines and saw minimal changes in the N:C ratio across the cancer cell lines (see S1 Table). The feature statistics from each measurement of each cell line were exported from IDEAS and imported into Jupyter Notebooks. Further post-processing involved the removal of images of cells which were analyzed as having nucleus diameters larger than that of the entire cell diameter. Lastly, the N:C ratio for the leftover cells was calculated. All data and post processing scripts can be found here.

Fig 2 — (a, c, e) Beeswarm plots and (b, d, f) density distributions display (a, b) cell, (c, d) nucleus, and (e, f) N:C ratio distribution across cell lines. Triple asterisks indicate a p-value of <0.001.

Results

Image flow cytometer

In this work, we interrogated four diverse cancer cell lines: OCI-AML-5, a blood cancer cell line; CAKI-2, a kidney cancer cell line, HT-29, a colon cancer cell line; and SK-BR-3, a breast cancer cell line. Initially, 43,237 OCI-AML-5, 19,699 CAKI-2, 32,907 HT-29, and 25,073 SK-BR-3 cells were imaged using the IFC within minutes. In our IFC gating process, we excluded out of focus cells, cell images containing alignment beads, and multiple or fragmented cells. We also removed objects analyzed in the IDEAS software that indicated a larger nucleus mask than cytoplasm mask. From this post-processing, we were able to analyze 5284 OCI-AML-5 cells, 1096 CAKI-2 cells, 6302 HT-29 cells, and 3159 SK-BR-3 cells. Table 1 and Fig 1B–1F provides a summary of the results of the IFC gating, analysis and post processing for each cell line and corresponding standard deviations. There is noticeable variability in the nuclear uptake of the DRAQ-5 as depicted in the 2^nd vertical panel of Fig 1C–1G. The variability of nuclear dyes has been studied and empirically determined as being cell cycle, cell-type, and concentration dependent [29–31]. To account for this variability in dye uptake, the fluorescent intensity gating process (Fig 1.a.IV) is individualized for each cell line. We also interrogated 158,069 MCF-10A non-malignant breast epithelial cells. Following the post-processing steps outlined above on the malignant cells, we were left with 21,396 cells that were DRAQ-5 positive but overestimated the N:C ratio of these cells. However, in looking at the IFC images, we noticed significant leakage of the nuclear dye out of the nucleus into the cell. Thus, we gated the fluorescent channel images based on their gradient root-mean-squared values to isolate cell images that were highly focused to distinguish between the nucleus and other parts of the cell (see Fig 1B). This left us with 1,109 cells MCF-10A cells. This modified template produced minimal changes to the N:C ratio when applied on all four malignant cell lines (see S1 Table) but did lower the analyze sample population. Overall, CAKI-2 cells had the largest diameter (24.5 ± 2.6 μm), followed by MCF-10A cells (19.4 ± 2.2 μm), SK-BR-3 cells (18.0 ± 3.7 μm), HT-29 cells (16.2 ± 1.8 μm), and lastly, OCI-AML-5 cells (12.3 ± 1.2 μm). The IFC measurements of cell diameter are in good agreement with published values of the OCI-AML-5 and HT-29 cell diameters that used the Coulter Counter [32], slightly larger than SK-BR-3 cell sizing using microfluidic cytometry [33], and in good agreement with published values of MCF-10As [34]. Moreover, our previous studies with MCF-7, PC-3 and MDA-MB-231 cells were all in good agreement with other validated techniques [25–27]. The nuclear diameters of these cell lines follow a similar trend, with CAKI-2 cells (15.6 ± 2.1 μm) having the largest nuclei, followed by SK-BR-3 cells (12.5 ± 2.1 μm), HT-29 cells (11.2 ± 1.3 μm), MCF-10A cells (10.1 ± 1.8 μm), and OCI-AML-5 cells (9.0 ± 1.1 μm). From the cell and nuclear diameters, the N:C ratio of each cell line was calculated. All cancerous cell lines, regardless of tissue origin, had similar N:C ratio values. OCI-AML-5 cells had the largest N:C ratio (0.73 ± 0.07), followed by SK-BR-3 cells (0.71 ± 0.13), followed by HT-29 cells (0.69 ± 0.07), CAKI-2 cells (0.64 ± 0.08). The non-malignant MCF-10A cells had the smallest N:C ratio (0.53 ± 0.11). In addition, given the standard deviation of the MCF-10A cells, their N:C ratios are also within range of published values [35]. Corresponding beeswarm plots and kernel density histograms of the size distributions for the cell diameter (Fig 2A and 2B), nucleus diameter (Fig 2C and 2D), and N:C ratio (Fig 2E and 2F) are shown in Fig 2.

Table 1. A summary of IFC measurements and corresponding standard deviations for the cell and nucleus diameter as well as the N:C ratio for each cell line.

Cell Line	OCI-AML-5 (n = 5284)	CAKI-2 (n = 1096)	HT-29 (n = 6302)	SK-BR-3 (n = 3159)	MCF-10A (n = 1109)
Cell Diameter [μm]	12.3 ± 1.2	24.5 ± 2.6	16.2 ± 1.8	18.0 ± 3.7	19.4 ± 2.2
Nucleus Diameter [μm]	9.0 ± 1.1	15.6 ± 2.1	11.2 ± 1.3	12.5 ± 2.1	10.1 ± 1.8
N:C	0.73 ± 0.07	0.64 ± 0.08	0.69 ± 0.07	0.71 ± 0.13	0.53 ± 0.11

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An ordinary one-way ANOVA with Tukey’s multiple comparisons test (alpha = 0.05) was used to test for significant differences between the means of the cell, nuclear, and N:C ratios between cell lines (GraphPad Prism v8.0, San Diego, USA). A statistically significant difference (p<0.001) was observed between all means between cell lines.

Discussion

The N:C ratio can be used as a histological metric in grading malignant disease in certain tissue types and cytologic specimens. In these biological samples, an enlarged nucleus has become a hallmark due to the abundance of chromatin present within malignant cells [1]. Currently, histology is the gold standard assessment method for the determination of the N:C ratio but it cannot be practically used when analyzing large populations of cells. Histology is advantageous when examining cohesive cells and tissue fragments, but it cannot be optimally used when analyzing large populations of cells. In addition, techniques which require a single cell suspension, such as IFC, would require special preparation techniques if using samples obtained from punch biopsies. These methods could in turn alter the cellular morphology and state of the cells and impact the resultant analysis. However, in many high-throughput clinical contexts, such as bodily fluid analysis for the detection of circulating tumor cells, minimal residual disease and hematological diseases, cytology would be cumbersome. Thus, an objective high-throughput technique to assess the N:C ratio would provide an approach that would provide this measurement more reliably and with larger sample datasets.

The drawbacks of histology to assess the N:C ratio and reported inaccuracies and inconsistencies in N:C assessments by morphologists and clinicians [9–11] have motivated the implementation of new techniques to determine the N:C ratio, including the use of computer vision [13], multi-photon microscopy [14–16], Cell-CT [17] and immunohistochemistry analysis techniques [18]. Rahmadwati et al. [13] applied k-means clustering to segment nuclei and cytoplasm from background and connective tissue to detect features indicative of normal tissue, pre-cancerous tissue, and malignant tissues in cervical cancer histology images to assess the N:C ratio. Morphological features were extracted from a region-of-interest (ROI) on sample histological images of normal tissue, pre-cancerous tissue, and malignant tissues. These extracted features were used to classify other histology slides as normal, pre-cancerous, or malignant. The technique is heavily ROI dependent and details regarding sample size and number of histological images used are not provided. Multi-photon microscopy techniques studied by Huang et al. [14, 15] suffered from low sample populations (n < 25) and thus, were not representative of the entire cell population and ineffective in high-throughput settings. A two-photon microscopy (TPM) technique was implemented by Su Lim et al. [16] to assess the nuclear area and N:C ratios in human colon tissues. Although the authors analyzed thousands of TPM images from ex vivo colon histological slices for 7 patients, their technique would not be feasible in a high-throughput context. Moreover, the technique is specific to colon cancer. The Cell-CT [17] device has been used to assess the nucleus-to-cytoplasmic ratio but has a lengthy imaging time. This poses a problem for live cell imaging as the cells may undergo apoptosis or other alterations during imaging. Lastly, Xu et al. [18] used Image Pro Plus 6 (Media Cybernetics Corporation, USA) to calculate the nuclear/cytoplasmic ratio of 70 pairs of gastric cancer tissues, that are positive for death domain associated protein 6 (Daxx), and adjacent normal tissues. Three microscope images at 400x magnification were obtained for each tissue sample and each image included at least 100 Daxx positive cells. This clinical study shows the application of the current gold standard, histological sectioning, combined with a computational histological analysis method. The subjectivity and low-throughput nature of histology is improving using digital pathology and computer-aided image analysis; however, IFC provides an alternative for high throughput analysis with large sample analysis populations measured in a short period of time and would be useful in high-throughput clinical contexts to assess the N:C ratio of certain types of cells.

Image flow cytometry boasts high-throughput and multiparametric abilities enabling the acquisition of morphological information of single cells. The lack of high throughput techniques available to analyze the N:C ratio provides a niche opportunity for this emerging cytometric method to be used in certain cell types. Although IFC does take 2D images of single cells, the high-throughput nature of IFC provides a more reliable estimate of the N:C ratio over an analysis of a single 2D histological slice. Moreover, histological sectioning analysis requires microscope images from sections of a slice with an unknown amount of stained positive cells. By combining the statistical and gating capabilities of flow cytometry with the imaging capabilities of bright-field microscopy, IFC provides an opportunity for the development of a statistically powerful analysis (thousands of cells) of the N:C ratio in cancerous and non-malignant cells. Here, we interrogated four cell lines of variable tissue origin (blood cancer, kidney cancer, colon cancer, and breast cancer) and a single non-malignant cell line (breast epithelium). Although the cell and nuclear diameters of each cancerous cell line differed (Fig 2) and statistical analysis revealed a significant difference between all their means, there is considerable overlap between their respective N:C ratios. However, we see a clear difference between malignant and non-malignant N:C ratios.

From Fig 2A and 2D, the measurements of cell diameter were consistent with the measurement of nuclear diameter over a range of cell sizes. This relationship between cell and nuclear size throughout the cell cycle is in line with what has been observed in the literature [36–38] and the future studies will examine the effect of the cell cycle [37] on the N:C ratio. In particular, the distribution of SK-BR-3 cell diameter found in Fig 2A and 2B does not show a Gaussian-like shape like the other cell lines did. The shape of the cell size distribution can be affected by the length of time the cell spends in each phase of the cell cycle. In this work however, the time in the S/G2/M phase is similar for all the cell lines studied (SKBR3–36%, HT-29–32%, CAKI-2–35%) [39–41]. Therefore, it is possible that the size variability of the SK-BR-3 cell line is inherent to this cell line.

To our knowledge, this high-throughput study is the first to measure the N:C ratio across malignant and non-malignant cells of different tissue origins. Our N:C results are consistent with international standards of cytopathology (Hang et al.’s [42] finding of an N:C ratio cutoff value of 0.5 for atypical urothelial cells, and McIntire et al.’s [43] finding of a N:C ratio cutoff value below 0.7 for high-grade urothelial carcinoma). Previous work by Rahmadwati et al. [13] and Huang et al. [14, 15] have shown that non-malignant cell types have N:C ratios between 0.2–0.4. Given the standard deviations of our non-malignant cell N:C ratios, our measurements fall within this range. To bolster our hypothesis related to the N:C distributions observed in our study, we plan on conducting a larger-scale study composed of multiple cell lines and their non-malignant counterparts. Additionally, since DRAQ-5 was the only nuclear dye examined in this study, the examination of the effects of various nuclear stains, their dye uptakes, and effects on the N:C ratio will be studied. However, we hypothesize that an alternate dye would not address the leakage problem as DRAQ5 has shown high nuclear localization in other studies [44]. Moreover, our future work will consider the effects of aneuploidy in the context of the N:C ratio. As many cell line subtypes can have varying nuclear size, shape, and complexity, their N:C ratio can be variable. It is beyond the capabilities of the currently presented technique to definitively state the cell subtype. However, a preliminary comparison between the N:C ratios of malignant and non-malignant cell lines does point towards a potential use for determining whether an individual cell is oncogenically transformed. Moreover, since this study focused on the presentation of a novel high-throughput technique to characterize the N:C ratio in multiple cell lines, the effects of aneuploidy were not considered.

IFC has some inherent disadvantages. Post-processing of the acquired cell images removed many images. Removal of images due to cells being out of focus, truncated cell boundaries, the presence of alignment beads in the field of view and all other factors resulted in the exclusion of up to 80–94% of acquired malignant cell images. Of the excluded images, 80–90% of the images were excluded during the gating step that determined whether the cell image was in focus. This exclusion was based on a cell edge gradient threshold of 60, commonly used in IFC gating analysis. The trypsinization of all cells before use in the IFC could have led to cytological damage to the cell membranes that caused their removal during the post-processing of the acquired IFC images In addition, we hypothesize that trypsin’s known damaging effects on the cell membrane [45] may have led to cell fragmentation that could have led to exclusion of affected cells during the post-processing of the acquired IFC images and account for the high cell loss in our IFC post-processing analysis. For example, the post-processing steps that gate single cell IFC images could have removed cell images that contained both the cell and its corresponding fragment due to trypsinization Initially, using the malignant cell line workflow, we noticed DRAQ-5 leakage out of the nucleus in the analyzed IFC images. We hypothesize that the extended immersion in the trypsin solution could have caused damage to the cell membrane [45], proteomic alterations [46], decrease in cellular protein expression [47], and damage to the nuclear proteins [48]. To account for this leakage, we added an extra gradient-root-mean-squared gate to isolate fluorescent cell images with sharp nuclear boundaries that were highly focused. This modified template produced minimal changes to the N:C ratio when applied on all four malignant cell lines (see S1 Table) but did lower the sample population that was analyzed. This subjective gating and masking approach is a limitation to imaging flow cytometry. Nevertheless, we are still able to reliably size thousands of cells. Modifications of this exclusion criteria could risk unfocused cells being used in proceeding gating steps and potentially impact the accuracy of our results. Moreover, cells with fragmented plasma membranes caused leaking of the DRAQ-5 into other parts of the cell which caused nuclear masks to be larger than cell masks. These cells were excluded in post-processing steps but demonstrates a limitation of the analysis technique to identify a stained nucleus if nuclear membrane fragmentation occurs. Similarly, the use of the “Diameter” feature to mask the cell or nucleus provides the diameter of a circle that has the same area as the masked cell or nucleus [28]. This step introduces the possibility of overestimating the nuclear diameter in cases of nuclear shape variability. Furthermore, although the gating strategies used in our analysis template were well-suited for the assessment of the N:C ratio, the potential user bias associated with the development of this template also introduces a subjective element in this approach. We have observed these common drawbacks to IFC in our previous work [49] and other groups have sought to improve the gating process [50, 51]. To eliminate these disadvantages of the IFC analysis workflow, our future work will focus on the use of computer vision and machine learning strategies to assess the N:C ratio. Here, no gating strategies are implemented to limit the loss of IFC images. Our group combined computer vision and machine learning strategies in the context of red blood cell storage lesions [52, 53]. We look to further incorporate techniques used by Doan et al. [54], Blasi et al. [55], and Hennig et al. [56] that use an unsupervised or weakly supervised, deep-learning method [57] for the assessment of the N:C ratio in both non-malignant and malignant cell lines. This would overcome drawbacks of IFC manual gating and user bias to provide an objective assessment of cell malignancy in a high-throughput context.

Conclusion

We present a high-throughput image flow cytometric assay to assess the cell diameter, nuclear diameter, and N:C ratio of four different malignant cell lines (OCI-AML-5—blood cancer, CAKI-2—kidney cancer, HT-29—colon cancer, and SK-BR-3—breast cancer) and a single non-malignant cell line (MCF-10A –breast epithelium). We observe that, although the cells had a wide range of cell and nuclear sizes, a general N:C ratio of ~0.6–0.7 is common to all interrogated cancer cell lines and an N:C ratio of 0.53 for the non-malignant cell line. Limitations of our analysis technique lie in the manual gating strategies used in the accompanying analysis software. Our future work will focus on the application of computer vision and machine learning techniques on IFC data to assess this metric in rare cell types and more non-malignant cells.

Supporting information

S1 Table. A summary of IFC measurements and corresponding standard deviations for the cell and nucleus diameter as well as the N:C ratio for each cell line using the modified IFC workflow on all non-malignant and malignant cell lines.

(DOCX)

Click here for additional data file.^{(15.1KB, docx)}

Acknowledgments

We gratefully acknowledge Michael Parsons for his key insights into the development of the gating template on the IDEAS software platform and his assistance with the Amnis ImageStream Imaging Flow Cytometer.

Data Availability

All data files are available on Zenodo (DOI: 10.5281/zenodo.4552851).

Funding Statement

This research was supported in part by the Natural Science and Engineering Research Council of Canada (https://www.nserc-crsng.gc.ca/index_eng.asp) Discovery Grant (RGPIN-2017-06486), the Canadian Foundation for Innovation (https://www.innovation.ca/) and the Ontario Ministry for Research and Innovation (Project #11525), and the Terry Fox Foundation (https://terryfox.org/, TFRI Project #1034) funding agencies. All grants were awarded to M.C.K. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0253439.r001

Decision Letter 0

Jeffrey Chalmers

9 Nov 2020

PONE-D-20-26034

An image-based flow cytometric approach to the assessment of the nucleus-to-cytoplasm ratio

PLOS ONE

Dear Dr. Sebastian,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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PLOS ONE

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: The manuscript reports a method for assessing N:C ratios in 4 cancer cell lines using DRAQ5 as a nuclear stain and imaging flow cytometry. The cell lines are exemplars to illustrate the approach. In addition, the N:C ratio needs to be compared with normal cells. This is especially relevant if the intent is to use this strategy to detect CTC; in this situation data of N:C for normal nucleated cells in blood (i.e. leucocytes) should be assessed.

If this work is merely to describe the method, then the precise biology of each cell line is not relevant. This comment is made as the details of the cell lines are not provided. Are any of the cell lines aneuploidy? Of particular note is the “AML cell” line which has no name or details. There are many subtypes of acute myeloid leukaemia and they can be heterogeneous with varying nuclear size, shape and complexity by subjective appearance by light microscopy. These cells are likely to have a broad range of N:C ratios. The N:C ratio needs to be compared with normal cells. This is especially relevant if the intent is to use this strategy to detect CTC; in this situation data of N:C for normal nucleated cells in blood (i.e. leucocytes) should be assessed. Also SK-BR3 is a breast cancer cell line derived from the pleural fluid, a metastatic site. As such it may not be representative of the primary tumour but may be aneuploid.

The loss of >80% of cells in the analysis raises questions over the methodology, instrument set up, validity of the data and applicability in practice. Did the cell loss occur pre-analytical as a consequence of the trypsin causing cytological damage to cell membranes or is the loss due to instrument factors? Can the authors address this high cell loss? The cell edge gradient/focus and clipped cells data shows a high level of flow core instability (>60 cell edge gradient). This reviewer does not agree with the authors comment that this is a standard limitation or operation of imaging flow cytometry. This needs to be addressed.

DRAQ-5 stain as demonstrated in Figure 1 shows variable intensity and quality between the 4 cell lines. Were DRAQ-5 titrations performed on the cell lines to standardise masking and stain intensity measurements? How did the “masking” strategy take this variability into account? Is the variable intensity of DRAQ-5 stain related to the cell line, stage of cell cycle or ploidy? Were any other fluorescent nuclear stains assessed? Would alternate stain have addressed the leakage issue? Figure 2f shows SK-BR-3 cells with bimodal distribution of N:C ratios. Is this variability due to stain intensity, cell proliferation or cell viability? Have any other DNA stains been used? Would these have addressed the leakage issue? How has nuclear complexity been taken into consideration when the nuclear diameter was measured? The cell cycle and nuclear content was acknowledged as influencing the N:C results. How did different stages of cell cycle affect nuclear measurements and subsequent N:C ratio?

The nuclear masks/fluorescent images shown in Figure 1B-E for the four cell lines highlight the potential for variability in nuclear content/configuration. The calculation of nuclear and cytoplasmic area, and by extension the N:C ratio is based on the “diameter” feature of the image masks for the nucleus and cell – calculations with “diameter” is only mathematically consistent if the objects have high circularity. Can the authors clarify the circularity feature/masks in the IDEAS software?

Reviewer #2: Sebastian et al. analyze the nuclear-to-cytoplasmic ratios of four cell lines derived from malignancies using image-based flow cytometry to demonstrate the use of this technology in measuring N/C ratios. The technology appears to effectively calculate N/C ratio histograms for a large number of events, which would not be feasible using traditional light microscopy. The average N/C ratios varied between the malignant cell lines, as expected, but were typically above 0.6 which is consistent with what has been found for high grade urothelial carcinoma. The study is concisely written and accessible and covers a topic that is of great interest in the field of cytopathology, both academically and commercially. Despite its simplistic design and results that are not immediately actionable, the study has great novelty and is impactful to the field of cytomorphology (and, because of that, digital pathology).

There are some issues that the authors should address:

1. The authors largely misunderstand the cited references 9-11. While enlarged N/C ratio is one morphologic feature of malignancy, in general, that is seen in both histology and cytology, the N/C ratio is most easily determined in cytologic specimens and not histology, and is currently mostly used in the study of urinary cytology. While the authors correctly cite the references 9-11, they incorrectly say on page 3 that this is the examination of histologic sections. In fact, this is not true, as cytology specimens are not sectioned and are not referred to as "histology" - the slides are fixed to a glass slide using any number of methods (primarily alcohol-based fixation or air fixation). Therefore, while the entire 3D structure of the cell can be examined by adjusting the microscope focus, the N/C ratio is typically calculated in one “Z-plane” in which both the nuclear contour and cytoplasmic contour appear most in focus. The authors should adjust their text to take this into account, and it would be relevant to comment on how the flow cytometric method determines which “Z-plane” contains the calculated N and C diameter values.

2. The authors use only malignant cell lines and it would have been nice to have used non-malignant control cells as well, even if not clonally expanded.

3. The authors should note that most epithelial malignancies taken directly from a patient will have cohesive cells and tissue fragments. To use this method would require breaking the cells apart, which may alter the cellular natural state, or would be biased towards individual cells, which typically are more pleomorphic than cells that remain in fragments. However, the method is immediately useful in malignancies which tend to be single cells (e.g. urothelial carcinoma and lymphoma/leukemia).

4. The authors should mention that N/C ratio is an important cytomorphologic feature of malignancy for many, but not all, tissue types. For instance, melanoma tends to have more cytoplasm and lower N/C ratios than urothelial carcinoma. Carcinomas can have greatly varied N/C ratios. However, it is true that a very high N/C ratio (>0.7) is usually not seen in benign cells.

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2021 Jun 24;16(6):e0253439. doi: 10.1371/journal.pone.0253439.r002

Author response to Decision Letter 0

20 Feb 2021

The response to Reviewer and Editor comments can be found in the attached file titled, "Sebastian_et_al_Rebuttals_SubmissionCopy.docx".

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Submitted filename: Sebastian_et_al_Rebuttals_SubmissionCopy.docx

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PLoS One. doi: 10.1371/journal.pone.0253439.r003

Decision Letter 1

Vincenzo L'Imperio

16 Apr 2021

PONE-D-20-26034R1

An image-based flow cytometric approach to the assessment of the nucleus-to-cytoplasm ratio

PLOS ONE

Dear Dr. Sebastian,

Due to the inavailability of one of the previous reviewers, we sent out your manuscript for a further evaluation by an additional independent reviewer which suggested to perform minor revision of the work.

Once the suggested modifications are addressed we will be glad to reconsider the paper for publication in the journal.

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Reviewers' comments:

Reviewer's Responses to Questions

Review Comments to the Author

Reviewer #2: I think the authors have satisfactorily answered the criticisms of the reviewers. Given their findings compared to now included normal cell control group, I think it is remarkable how closely the N/C cut-offs correlate with the findings in two studies of urothelial carcinoma in which image-based morphometric analysis was used. While I do not think the authors necessarily need to further modify their manuscript, the findings do, in general, support the authors' findings. And, if the authors wish to pursue this area further, discussion in these manuscripts may provide valuable:

1. Digital image analysis supports a nuclear-to-cytoplasmic ratio cutoff value of 0.5 for atypical urothelial cells. PMID: 28581671 DOI: 10.1002/cncy.21883

2. Digital image analysis supports a nuclear-to-cytoplasmic ratio cutoff value below 0.7 for positive for high-grade urothelial carcinoma and suspicious for high-grade urothelial carcinoma in urine cytology specimens. PMID: 30395388 DOI: 10.1002/cncy.22061

Reviewer #3: The Authors describe the application of IFC to the measurement of the N:C ratio in one benign and four malignant human cell lines.

The methods and their description, as well as the overall manuscript style and language, are irreproachable.

However, my impression is that the study suffers from the absence of a pathologist/physician among the Authors (both in the overall design and in the manuscript itself).

Let us consider the main ideas of the study separately:

1. IFC has potential as a technique to measure N:C

The Authors convincingly show that IFC can be used to measure the N:C ratio of a population of cells. This idea is original and supported by the study design and data.

However, the practical applications of this are arguable because histology (especially if empowered by computer-aided image analysis) might be better, considering that it is already routinely performed on all human cancers and does not require other tools (like an IFC).

2. the N:C ratio (measured by IFC) can be used as a discriminator of malignancy

In my opinion, this premise is flawed. It will be discussed below.

--------

56: "Clinicians have identified an enlarged nucleus as one of the most prevalent characteristics of malignant cells"

An enlarged nucleus is definitely a characteristic of some neoplasms. However, it is neither sensitive nor specific. There are benign cells with huge nuclei (think symplastic leiomyoma) and malignant cells with tiny nuclei (signet ring cell cancers, foamy prostate cancer). Similarly, the N:C ratio is increased in most cancers, but again it is neither sensitive nor specific. There are benign cells with alarmingly high N:C ratios (lymphocytes) and malignant cells with very low N:C ratios (again, signet ring cells). Furthermore, some neoplasms are by definition cytologically indistinguishable from their benign counterparts (and thus they share an identical N:C ratio). This is the case of follicular thyroid adenoma/carcinoma and several other endocrine neoplasms.

211: "The N:C ratio is widely used as a histological metric in staging malignant disease in most tissue types". This is not true, as far as I'm aware. Histological grading (not staging) of cancers depends on the histotype, but only rarely does the N:C ratio make an appearance in grading criteria. For example, prostate cancer is graded based on architecture alone; endometrial, colorectal, and lung cancers are graded mostly based on architecture; even in neoplasms in which the N:C ratio is somehow considered in grading (urothelium, breast), it is just one of several criteria.

212: "An enlarged nucleus has become a hallmark in tumor staging and grading due to an abundance of chromatin present within malignant cells". Staging and grading are two distinct things. Grading has been discussed previously. Staging of most if not all human cancers uses the AJCC TNM system which does not take the N:C ratio into account at all.

Minor points:

- 48: the Authors seem to suggest that histology as the gold standard technique to diagnose malignancy should be replaced by other, high-throughput, techniques. This is arguable, but not supported by the rest of the paper.

- 240: what's TPM?

- 250: histological sectioning is indeed tedious, but it is performed by routine on all human cancers, so the added cost would be minimal.

- Is the N:C ratio calculated as a ratio of diameters, areas or volumes?

- 59: Measuring the N:C ratio by visual approximation on histological images is indeed error-prone and operator-dependent. However, computer-aided image analysis can overcome this problem.

- 214: "Currently, histology is the gold standard assessment method for the determination of the N:C ratio but it cannot be practically used when analyzing large populations of cells". Partly true: digital pathology and computer-aided image analysis are overcoming this problem. The Authors themselves disprove this statement at line 225.

In conclusion, IFC is an interesting and promising technique, and it can surely be used to measure the N:C ratio of cells (as this manuscript shows). However, the idea of diagnosing malignancy based on the N:C ratio (measured by IFC or otherwise) seems flawed. The study would be better reorganized by focusing on the N:C measurement by IFC as the main finding and discarding any implications on the diagnosis of malignancy.

**********

PLoS One. 2021 Jun 24;16(6):e0253439. doi: 10.1371/journal.pone.0253439.r004

Author response to Decision Letter 1

11 May 2021

All Review rebuttals can be found in the document "Sebastian_et_al_Rebuttals_2_SubmissionCopy.docx".

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PLoS One. doi: 10.1371/journal.pone.0253439.r005

Decision Letter 2

Vincenzo L'Imperio

26 May 2021

PONE-D-20-26034R2

An image-based flow cytometric approach to the assessment of the nucleus-to-cytoplasm ratio

PLOS ONE

Dear Dr. Sebastian,

After a re-evaluation of the revised manuscript, one of the reviewer raised additional observations that could be addressed to further improve the quality of the paper. We'll be glad to receive the new version of the work after the corrections suggested for the final decision on approval.

Please submit your revised manuscript by Jul 10 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
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We look forward to receiving your revised manuscript.

Kind regards,

Vincenzo L'Imperio

Academic Editor

PLOS ONE

Journal Requirements:

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Reviewers' comments:

Reviewer #2: The authors have appropriately addressed all comments. However, I want to make a few corrections in their response and modifications.

- For the new statement “Our N:C results are consistent with international standards of cytopathology (such as Zhang et al.’s (10) finding of an N:C ratio ≥0.7 as malignant, Hang et al.’s (42) finding of an N:C ratio cutoff value of 0.5 for atypical urothelial cells, and McIntire et al.’s (43) finding of a N:C ratio cutoff value below 0.7 for high-grade urothelial carcinoma).” please remove reference 10 here. Reference 10 is correctly cited earlier in the manuscript as it studied interobserver variability, but it did not study the impact of N/C ratio on determining whether cells were malignant or not.

- Regarding the comments of reviewer #3, I agree that the N/C ratio is not in general a standard used in histopathologic examination for grading malignancies. It is true that it plays a role in some types of malignancy as increased N/C ratio is one of many features used to assess a cell for cytologic atypia. I bring this up because the authors seem to focus mostly on N/C ratio in tissue sections when the assessment is more readily done in cytologic specimens, and in cytology the only formal system that requires an N/C ratio assessment is The Paris System for urine specimens. So, while the N/C ratio is one of many cytologic features that can help diagnose malignancy or grade/differentiation of a malignancy, it depends on the specimen type and the cancer type. I would recommend double checking the manuscript to ensure the introduction and discussion is consistent with this idea. In part, because the N/C ratio can be difficult to assess in both histology and cytology, is is important that the authors have shown a way to objectively measure the N/C ratio and correlate ratios with malignant vs. benign cells. This can help show the degree to which N/C ratio variations are associated with malignancy, at least in certain cell types, even if no single feature can be used, in most cases, to determine whether a cell is malignant or benign.

Reviewer #3: All comments have been addressed in a satisfactory manner.

State-of-the-art high-throughput whole slide scanners coupled with appropriate software are already usable to get N:C data in high-throughput contexts; however I have to agree that until these become ordinary, they represent an extra cost (not unlike an IFC).

**********

PLoS One. 2021 Jun 24;16(6):e0253439. doi: 10.1371/journal.pone.0253439.r006

Author response to Decision Letter 2

26 May 2021

The Reviewer replies is in a document titled, "Sebastian_et_al_Rebuttals_3_SubmissionCopy".

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Submitted filename: Sebastian_et_al_Rebuttals_3_SubmissionCopy.docx

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PLoS One. doi: 10.1371/journal.pone.0253439.r007

Decision Letter 3

Vincenzo L'Imperio

7 Jun 2021

An image-based flow cytometric approach to the assessment of the nucleus-to-cytoplasm ratio

PONE-D-20-26034R3

Dear Dr. Sebastian,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Vincenzo L'Imperio

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer #2: All comments have been addressed

PLoS One. doi: 10.1371/journal.pone.0253439.r008

Acceptance letter

Vincenzo L'Imperio

16 Jun 2021

PONE-D-20-26034R3

An image-based flow cytometric approach to the assessment of the nucleus-to-cytoplasm ratio

Dear Dr. Sebastian:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Vincenzo L'Imperio

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

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Data Availability Statement

All data files are available on Zenodo (DOI: 10.5281/zenodo.4552851).

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PERMALINK

An image-based flow cytometric approach to the assessment of the nucleus-to-cytoplasm ratio

Joseph A Sebastian

Michael J Moore

Elizabeth S L Berndl

Michael C Kolios

Roles

Abstract

Introduction

Methods

Cell preparation

Image flow cytometer operating parameters and post-processing

Fig 1. IFC analysis workflow and representative cells.

Fig 2. Cell diameter, nuclear diameter, and N:C ratio distribution.

Results

Image flow cytometer

Table 1. A summary of IFC measurements and corresponding standard deviations for the cell and nucleus diameter as well as the N:C ratio for each cell line.

Discussion

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Jeffrey Chalmers

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Author response to Decision Letter 0

Decision Letter 1

Vincenzo L'Imperio

Roles

Author response to Decision Letter 1

Decision Letter 2

Vincenzo L'Imperio

Roles

Author response to Decision Letter 2

Decision Letter 3

Vincenzo L'Imperio

Roles

Acceptance letter

Vincenzo L'Imperio

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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RESOURCES

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