Fig 1.
ldbFUS transducer and hydrophone (Sonic Concepts H107 & Y107) are fixed in a plastic coupling cone filled with degassed water and sealed with a thin membrane. Ultrasound gel between membrane & tumor ensure good sonic contact. Focal region of ldbFUS transducer is ~5mm diameter & 15mm long; centered below the coupling membrane and within the tumor.
Fig 2.
Relaxation rate is linear in Fe labeled NK cell concentration.
Ferumoxytol labeled NK cells were suspended in both 26% Ficoll and 1% agar solutions and R2* determined at 7T using 2D MGE protocol (mean±SD). There is no significant difference between fitted slopes and intercepts of Fe-NK suspensions in 26% Ficoll and 1% agar solutions.
Fig 3.
R2* maps and tumor histograms monitor changes in NK cell concentration.
(A) Representative parametric R2* images (axial slices) of an NSG mouse bearing bilateral flank LS-174T tumors before (pre), 1hr, 6hr, and 24hr post ldbFUS/Fe-NK administration. ldbFUS was induced at peak rarefication pressure of 0.50MPa. Yellow outlines show tumor regions of interest. Note that at some imaging time points left and right tumors were not in the same field of view–thus left & right sides (dashed line) may be from slightly different axial slices. Units in sec-1. (B) Histogram showing relative frequency R2* distributions of whole tumors from (A). The histogram shifts to increasing values of R2* with time in (+)ldbFUS tumor.
Fig 4.
Boxplots of ΔR2* for LS-174T tumors without (-) and treated with (+) ldbFUS and non-tumoral back muscle regions in NSG mice that received either low power (0.25MPa, left) or higher power (0.50MPa, right) ldbFUS.
Thick center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend to 1.5 times the interquartile range; diamond-shaped markers represent sample means. A Kruskal Wallis H test showed that there was a statistically significant difference in ΔR2* between the four groups (p<0.00001). Post hoc comparisons (pre vs. post or (-) vs. (+) ldbFUS) that showed significant differences are indicated by *p<0.05 and **p<0.01. NS = no significant difference.
Fig 5.
Boxplots of Apparent Diffusion Coefficient (ADC) values for a subset of LS-174T tumors (n = 12) without (-) and treated with (+) ldbFUS/0.50MPa in NSG mice.
Thick center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend to 1.5 times the interquartile range; diamond-shaped markers represent sample means. There were no significant changes in ADC values between ldbFUS treated and non treated tumors and across time points.
Fig 6.
NK cell concentration in tumors determined from ΔR2*.
Although NK cell tumor distributions are heterogeneous as seen in Fig 3, R2* geometric means were obtained by fitting the R2* histogram to a lognormal distribution. NK cell concentrations (106/ml, mean±SD) in tumor tissue obtained from ΔR2* determination and the linear relationship between R2* and NK cell concentration shown in Fig 2. Planned comparisons were performed using the Student’s t-test. Significant differences are indicated by *p<0.05 and **p<0.01.
Fig 7.
Anti-CD56-HRP plus Prussian Blue staining of spleen and tumor regions show accumulation of NK cells.
Spleen (A) and tumor regions (B, C) are from an NSG mouse. (B) Tumor region receiving ldbFUS (0.5MPa) and (C) tumor contralateral to B. (D) Tumor region from an NSG that received no ldbFUS treatment. Scale bar = 50 μm. Arrows show greater number of colocalization of positive CD56 (brown) and positive iron (blue) staining from ldbFUS administered tumor than tumors without treatment.
Fig 8.
Quantitation of % NK cells from fluorescent staining of tumors from an NSG mouse.
(A) Fluorescent NK cells (CD56, red) and DAPI (blue) staining of tumors from an NSG mouse: upper panel from tumor administered ldbFUS/0.50 MPa; lower panel from contralateral tumor that was not treated. Scale bar = 50 μm. (B) A sample section with positive-NK-CSFE (green) in tumor tissue (DAPI, blue). Scale bar = 50 μm. (C) Mean±SD Percent % NK cells determined from CD56 and CSFE fluorescent staining. % NK cells = number of NK cells / total number of cells (determined from DAPI stain). Planned comparisons using t-test showed a significant difference between (-)ldbFUS and (+)ldbFUS, **p<0.01.