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. 2012 Jul 18:2:72.
doi: 10.3389/fonc.2012.00072. eCollection 2012.

Optical quantification of cellular mass, volume, and density of circulating tumor cells identified in an ovarian cancer patient

Kevin G Phillips  1 , Carmen Ruiz Velasco, Julia Li, Anand Kolatkar, Madelyn Luttgen, Kelly Bethel, Bridgette Duggan, Peter Kuhn, Owen J T McCarty
Affiliations

Optical quantification of cellular mass, volume, and density of circulating tumor cells identified in an ovarian cancer patient

Kevin G Phillips et al. Front Oncol. .

Abstract

Clinical studies have demonstrated that circulating tumor cells (CTCs) are present in the blood of cancer patients with known metastatic disease across the major types of epithelial malignancies. Recent studies have shown that the concentration of CTCs in the blood is prognostic of overall survival in breast, prostate, colorectal, and non-small cell lung cancer. This study characterizes CTCs identified using the high-definition (HD)-CTC assay in an ovarian cancer patient with stage IIIC disease. We characterized the physical properties of 31 HD-CTCs and 50 normal leukocytes from a single blood draw taken just prior to the initial debulking surgery. We utilized a non-interferometric quantitative phase microscopy technique using brightfield imagery to measure cellular dry mass. Next we used a quantitative differential interference contrast microscopy technique to measure cellular volume. These techniques were combined to determine cellular dry mass density. We found that HD-CTCs were more massive than leukocytes: 33.6 ± 3.2 pg (HD-CTC) compared to 18.7 ± 0.6 pg (leukocytes), p < 0.001; had greater volumes: 518.3 ± 24.5 fL (HD-CTC) compared to 230.9 ± 78.5 fL (leukocyte), p < 0.001; and possessed a decreased dry mass density with respect to leukocytes: 0.065 ± 0.006 pg/fL (HD-CTC) compared to 0.085 ± 0.004 pg/fL (leukocyte), p < 0.006. Quantification of HD-CTC dry mass content and volume provide key insights into the fluid dynamics of cancer, and may provide the rationale for strategies to isolate, monitor or target CTCs based on their physical properties. The parameters reported here can also be incorporated into blood cell flow models to better understand metastasis.

Keywords: cellular density; cellular mass; cellular volume; circulating tumor cell; differential interference contrast; ovarian cancer; quantitative phase microscopy.

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Figures

Figure 1
Figure 1
Transport of intensity based quantitative phase microscopy validation over two orders of magnitude. (A,C,E) Brightfield imagery of polystyrene spheres (n = 1.597, λ = 540 nm) suspended in fluoromount G (n = 1.4) with diameters of 0.11, 0.95, and 4.8 tm, respectively. (B,D,F) Corresponding transport of intensity based quantitative phase maps. (G,H,I) demonstrate theoretical phase profiles (dashed) for each polystyrene sphere with corresponding data (circles) overlaid.
Figure 2
Figure 2
HD-CTC identification of ovarian cancer CTCs and corresponding differential interference contrast (DIC) imagery. (A,C) Immunofluorescent identification of HD-CTCs: CK+CD45−DAPI+ and peripheral leukocytes: CK−CD45+DAPI+ (B,D) corresponding differential interference contrast images of the same fields.
Figure 3
Figure 3
Quantitative phase microscopy of circulating tumor cells; comparison to differential interference contrast (DIC) and brightfield microscopy. (A,D) Differential interference contrast imagery of CK+CD45-DAPI+ HD-CTC5. (B,E) Corresponding bright field images. (C,F) Quantitative phase microscopy images determined from the transport of intensity equation analysis applied to brightfield through-focus imagery.
Figure 4
Figure 4
Quantification of biophysical properties of ovarian cancer HD-CTCs. (A) Scatter plot of cellular dry mass in pg (abscissa) versus cellular volume in fL (ordinate). Quantitative phase microscopy based mass measurements and differential interference contrast based volume measurements were uncorrelated. (B) Density box and whisker plot, (C) mass box and whisker, (D) volume box and whisker, (E) area box whisker. *Denotes p < 0.05 with respect to CTCs.
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