Supplemental Materials

Transcription

1 1 1 Supplemental Materials Figure S1: Schematic diagram of the BDI instrument. The BDI system is a short-coherence Mach Zender interferometer with digital holographic acquisition and reconstruction. The three-dimensional capabilities are provided by coherence-gated detection that is equivalent to laser ranging using time-offlight detection of backscattered light. Low-coherence light is provided by a superluminescent diode (SLD). A polarizing beam splitter (PBS) separates light from the SLD into an object beam (directed downwards in the figure) and a reference beam (directed to the right of the figure). The reference beam passes through a computer-controlled delay line, while the object beam is projected onto the tumor sample. Backscattered light from the tumor sample is Fourier transformed by the lenses (L1 and L2) and interferes with the reference beam at the Fourier plane (FP) of the object beam. Interference of the object beam with reference beam produces a speckle pattern which changes over time as a function of dynamic subcellular motion within the tumor sample. The speckle pattern is captured by a charge-coupled device (CCD) camera as a digital hologram for subsequent image reconstruction and analysis. BS = Beam splitter, IP = Image plane. 16

2 Figure S2 Graphical BDI data outputs and their interpretation. The two formats for visual representation of BDI data are motility contrast images (Figure 1) and drug response spectrograms (Figure 3 and S2). Motility contrast images are spatial maps of temporal fluctuations in aggregate subcellular motion at a fixed depth within the tumor. Figure 1 depicts four motility contrast images, acquired either before or after ex vivo doxorubicin treatment, of tumor biopsy samples from two dogs with NHL. The roughly circular structures in each image represent whole living tumor samples measuring approximately 1 mm in diameter. A false-color scale bar at the right-hand side of the panel describes change in aggregate motion of each pixel in the images averaged over time. Red pixels are associated with higher subcellular motion, whereas blue pixels are associated with lower subcellular motion. In these images, a tumor from a non-responder dog in the top two panels shows a higher level of baseline subcellular motion, and less reduction in post-treatment subcellular motion, than does a tumor from a responder dog in the bottom two panels. Drug response spectrograms are time-frequency representations of the change in fluctuation spectral content within a tumor sample in response to an applied drug. Figure S2 depicts a drug response spectrogram from a canine NHL biopsy sample which was treated with doxorubicin ex vivo. Time is plotted on the y-axis and frequency of measured motion is plotted on the x-axis. The dashed vertical lines divide the spectrogram into high-, mid-, and low-frequency ranges. The solid horizontal line denotes when the biopsy sample was treated with doxorubicin ex vivo. Motion is an inextricable feature of the numerous physiologic activities performed by the cell, although the frequency of motion at which each of these activities occurs varies. BDI detects diverse subcellular motions occurring across a frequency range of Hz. Large-scale motion affecting the morphology of the entire cell, such as membrane blebbing during apoptosis, occurs at low frequencies ( Hz). Intermediate scale motion, such as that associated with membrane transport processes, occurs at the middle frequency range ( Hz). Small scale motion, such as that associated with internal organelles, occurs at higher frequencies ( Hz). As in figure 1, the false-color scale bar at the right-hand side of the figure describes change in motion over time, with red pixels corresponding to increased subcellular motion, and blue pixels

3 corresponding to decreased subcellular motion. In this image, it is apparent that ex vivo treatment with doxorubicin elicited an increase in low-frequency motion and suppression of mid- and high-frequency motion within the tumor cells comprising the biopsy sample. Very large scale cell motions, such as those produced by cell migration during invasion or metastasis, do not contribute to drug response spectrograms because these processes are associated with Doppler frequencies below the lowest detection frequency (0.01 Hz) measured by BDI Figure S3: BDI-measured biomarkers of subcellular motion and their interpretation. Subcellular motion can be measured in many ways with BDI. By analyzing previously generated motility contrast images and drug response spectrograms, we have characterized six biomarkers of dynamic subcellular motion: NSD, DNSD, FULLSPEC, MEM, APOP, and BLUETAIL.

4 Review of figures 1 and 2 in the main article will provide an understanding of the NSD and DNSD biomarkers. NSD reflects pixel intensity in motility contrast images averaged over time, describing aggregate subcellular motion within a tumor sample. In figure 1, the greater number and intensity of red pixels in the top left image indicates that this tumor has a higher baseline NSD value than the tumor in the bottom left image. In principle, NSD can be measured across any span of time depicted along the x-axis in figure 2. However, for the purposes of this work, the biomarker value measured as NSD refers only to pixel intensity averaged over the baseline time span prior to doxorubicin application. Thus, the NSD biomarker is a measure of baseline aggregate subcellular motion in untreated tumor samples, and is measured in the span between time = 0 and the time of doxorubicin application in figure 2. In contrast, the DNSD biomarker describes the change, relative to baseline, in pixel intensity in motility contrast images caused by a tumor s response to doxorubicin. In figure 1, the tumor in the bottom two images displays a greater reduction in aggregate subcellular motion following ex vivo drug treatment, and therefore has a greater DNSD value, than the tumor in the top two images. In figure 2, the time span over which DNSD is measured extends from the time of doxorubicin application to the far right edge of the x-axis. The FULLSPEC, MEM, APOP, and BLUETAIL biomarkers are measured from drug response spectrograms. Figure S3A shows an example of a drug response spectrogram with a large negative FULLSPEC value. This spectrogram pertains to a biopsy obtained from a study dog with NHL, and demonstrates marked suppression of cell motion at all frequencies following ex vivo treatment with doxorubicin given at time = 0 (denoted by the dashed horizontal line on the spectrogram).

5 MEM describes changes in motion apparent within the middle frequency range of drug response spectrograms. Figure S3B shows an example of a drug response spectrogram with a large MEM value, which tends to correlate with resistance of a tumor to a particular drug. 80

6 APOP describes changes in motion occurring at both high (>1 Hz) and low (<0.1 Hz) frequencies on drug response spectrograms. Figure S3C shows an example of a drug response spectrogram with a large negative APOP value, in which low- and high-frequency motion is increased, but mid-frequency motion is suppressed. Spectrograms with this appearance have been correlated previously with tumor cell apoptosis following ex vivo drug treatment. 88

7 BLUETAIL denotes the extent to which reduction in motion is apparent at any frequency, across all of the samples obtained from each dog s tumor, following ex vivo drug treatment. For example, the spectrogram in figure S3A has both a large negative FULLSPEC value and a large positive BLUETAIL value since suppression of motion is present across the entire spectrogram. The spectrogram in figure S3C, by comparison, has a somewhat large positive BLUETAIL value produced by the marked suppression of motion in the middle frequency range. However, this spectrogram does not have as negative a FULLSPEC value as that in figure S3A since suppression of motion in the low and high frequency ranges is not as pronounced in the former spectrogram as it is in the latter. A further example of the derivation of the BLUETAIL biomarker is provided in figure S Figure S4: Intratumoral heterogeneity in subcellular motion demonstrated by BDI. Several tumor biopsy samples from each dog in this study were analyzed using BDI, revealing considerable heterogeneity in subcellular motion throughout each dog s tumor, both prior to and following ex vivo doxorubicin treatment. Figure S4 shows drug response spectrograms generated from each of 12 processed tumor

8 biopsy samples from a single dog with NHL. Doxorubicin (10 M) was applied at time = 0 (thin blue horizontal line toward the bottom of each spectrogram). BDI generated a unique drug response spectrogram from each of the 12 tumor biopsy samples from this dog. There is marked variability in spectral data obtained by imaging each biopsy sample. To account for this intratumoral heterogeneity, data from all 12 spectrograms were averaged (larger spectrogram on the right side of the figure) for the purposes of statistical analysis Figure S4 further provides a further example for interpreting the BLUETAIL biomarker. BLUETAIL denotes the extent to which reduction in motion is apparent at any frequency, across all of the samples obtained from each dog s tumor, following ex vivo drug treatment. The samples in figure S4 have a high BLUETAIL value caused by the strong motion-suppressive responses in several of the spectrograms Supplemental Table S1: Measured values of dynamic motility biomarkers from tumor biopsy samples in 10 dogs with NHL. The values are reported in relative units, which reflect specific patterns of change in

9 relative pixel intensity across a motility contrast image or drug response spectrogram following ex vivo application of doxorubicin to a tumor sample. SEM = Standard error of the mean. Responder dogs Dog APOP FULLSPEC MEM BLUETAIL DNSD NSD Mean SEM Non-responder dogs Dog APOP FULLSPEC MEM BLUETAIL DNSD NSD Mean SEM