Three-dimensional nucleus-to-cytoplasm ratios provide better discrimination of normal and lung adenocarcinoma cells than in two dimensions

Abstract

We acquired multiphoton images of normal and lung adenocarcinoma cell lines in three dimensions. Image stacks of the cells were then processed to obtain nucleusto-cytoplasm (N/C) ratios in two and three dimensions. While N/C ratios in three dimensions can be unambiguously determined from the volumetric ratios of the nucleus and cytoplasm, two-dimensional (2-D) N/C can vary depending on the axial plane selected for N/C ratio determination. We determined 2-D N/C ratios from three criteria: (1) axial position at which the nuclear area is the largest; (2) the largest 2-D N/C ratio value; and (3) axial position at the midpoint of nuclear axial position. We found that different definitions of 2-D N/C ratio will significantly affect its value. Furthermore, in general, larger variance was found in 2-D rather than three-dimensional (3-D) N/C ratios. Lack of ambiguity in definition and reduced variance suggest that 3-D N/C ratio is a better parameter for characterizing tumor cells in the clinical setting. © The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

Circulating Tumor Cell Subtypes and T-cell Populations as Prognostic Biomarkers to Combination Immunotherapy in Patients with Metastatic Genitourinary Cancer

“…Subtypes B and D were associated with worse OS not specific to UC. There was no distinct urothelial signature found in the pattern of morphologic subtype expression compared to the other malignancies in our cohort, supporting the pan-cancer theory that certain cellular features are negatively prognostic across cancer types (23). Interestingly, subtype E which contains CTC clusters was rarely detected.…”

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

“…The drawbacks of histology to assess the N:C ratio and reported inaccuracies and inconsistencies in N:C assessments by morphologists and clinicians [9][10][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][15][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.…”

“…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.…”

“…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.…”

An image-based flow cytometric approach to the assessment of the nucleus-to-cytoplasm 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][14][15][16][17][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.…”