STUDY RATIONALE: The demonstrated ability of MCT to accurately assess tumor xenografts may establish it as a reliable technique for patient tumor stratification based on predicted response to therapy, which could enable a treatment selection based on the personal needs of an individual patient. This study is designed to assess MCT as an assay for predicting chemosensitivity to treatment with chemotherapy agents routinely used in the neoadjuvant and adjuvant treatment of breast cancer. If positive, the results of this study will provide the basis for expanded clinical trials and use of MCT in therapy selection.
BACKGROUND: Live cell imaging has become the standard for high-content analysis and drug discovery applications. The most common assays on live cells include viability, proliferation and cytotoxicity assays as cellular physiology and function are measured responding to applied perturbations of xenobiotics. Cellular and tissue viability assays are typically measured using exogenous vital dyes as biomarkers of membrane integrity or cellular metabolic activity. However, dyes are invasive, potentially toxic, and often require fixing of the tissue or permeabilization of the membranes \[1, 2\]. Furthermore, the common format of high content analysis and flow cytometry requires isolated cells, or cells distributed on flat hard surfaces. Isolated cells lack many of the biologically-relevant intercellular connections and communications that are hallmarks of healthy tissue \[3, 4\], and flattened cells on plates have pathological shapes and anisotropic cellular adhesions \[5\].
Discovery of technology that can predict response to cancer therapy is an urgent priority. While many technologies exist to evaluate early response to drugs ex vivo, the need to perform viability, cytotoxicity and proliferation assays in three-dimensional tissue or culture has become increasingly urgent \[6, 7\], as drug response in 2D is often not the same as drug response in biologically-relevant three dimensional culture. This is in part because genomic profiles are not preserved in monolayer cultures \[8-10\]. There have been several studies that have tracked the expression of genes associated with cell survival, proliferation, differentiation and resistance to therapy that are expressed differently in 2D cultures relative to three-dimensional culture. For example, cell lines of epithelial ovarian cancer \[11, 12\], hepatocellular carcinoma \[13-15\] or colon cancer \[16\] display expression profiles more like those from tumor tissues when measured in three-dimensional culture, but not when grown in 2D. In addition, the three-dimensional environment of 3D culture presents different pharmacokinetics than 2D monolayer culture and produce differences in cancer drug sensitivities \[17-20\]. Finally, most current technologies rely on destructive end-point assessment, preventing meaningful longitudinal observation of therapy response over time.
One of the main challenges to migrating drug-response assays to the third and fourth dimensions has been to find a means to extract vital information from deep (up to a millimeter) inside living tissue. Tissue is translucent and light can propagate diffusively many centimeters. Furthermore, the dynamic motions of living cells cause dynamic light scattering that produces phase fluctuations on the scattered light fields that can be measured as dynamic speckle in diffusely reflected light from tissue. This is the basis of diffusing wave spectroscopy (DWS) \[21, 22\] and diffusion correlation spectroscopy (DCS) \[23-26\], but these techniques lack depth resolution. A powerful tool in the characterization of light propagation in tissues is the use of interferometry \[27\]. Interferometric detection is the underlying process in optical coherence tomography (OCT) \[28-31\], which is a point-scanning technique that suppresses speckle \[32-34\], although speckle decorrelation in OCT data can provide similar information as provided by DCS. This has been used to measure intracellular rheology \[35\] and to find dynamic signatures of apoptosis \[36\]. Transport also can be detected at cellular resolution using phase contrast microscopy \[38\], but this approach cannot be used in thick tissues.
Dr. Nolte and colleagues have developed volumetrically-resolved tissue dynamic imaging that uses the advantages of depth selectivity from low-coherence interferometry, combined with high speckle contrast in broad-illumination digital holography. The technique is called Motility Contrast Tomography (MCT) and uses low-coherence digital holography to penetrate up to 1 millimeter into living tissue to measure speckle dynamics from light scattering from dynamic motion in living cells \[37\]. It was previously applied as a cytotoxicity assay to study the efficacy of anti-mitotic drugs \[40\]. In essence, the technology works by profiling the 'movement' of cellular organelles. Specific changes in organelle motion are detectable very early in cells undergoing response to chemotherapy treatment, and may be usable as an early predictor of chemotherapy response.
MCT is based on optical coherence imaging (OCI) \[38\]. OCI is a full-field short-coherence holography \[39\] that collects backscattered speckle. With the help of coherence gating, OCI can optically section tissue up to 1 mm deep. MCT specifically uses intracellular motion as the endogenous contrast to characterize submicron subcellular motion inside three-dimensional living tissue \[42\].
Figure 1 shows the holographic recording principle of MCT. After calibration, the short coherence light is first divided into an object beam and a reference beam. The object beam hits the living tissue sample, and backscattered speckles from the tissue are collected by the lens L1. The living tissue sample locates at the focal plane of the lens L1, so L1 also performs an optical Fourier transform of the backscattered light. The charge coupled device (CCD) locates at the other focal plane of L1, so it captures the Fourier transformed scattering light from the tissue. The reference beam is controlled by a delay stage (not shown in the figure) to adjust the path length of the reference beam to perform a zero-path match between the object and reference beams. The beam splitter combines both beams and because they are zero-path matched, they interfere at the CCD plane. The reference beam is tilted by 20° in an off-axis configuration, and the spacing of the interference fringes (Λ) is 3 pixels (24 μm). The speckle size (aspec) is adjusted to be 3 fringes wide (70 μm). Additional details about the experimental setup can be found in reference \[41\].
Fig.1. The principle of MCT on multicellular tumor spheroids. The biological sample is located at the image plane of lens L1. The back scattered light from the sample is Fourier transformed by L1 and interfered with reference beam on the CCD chip. The speckle hologram is recorded on the Fourier plane with a 20 crossing angel with the reference beam. Examples of a) Raw digital hologram; b) reconstructed image; c) MCI image. O.A.: optical axis; I.P.: image plane; L1: lens; BS: beam splitter; CCD: charge coupled device.
EX VIVO CANCER CHEMOSENSITIVITY ANALYSIS MCT was previously applied to study the efficacy of anti-mitotic drugs using multicellular tumor spheroids \[40\]. When applying MCT to tumor xenografts, it is also capable of showing a significantly different response between two cell lines under cisplatin. After applying the drug, the normalized standard deviation (NSD) value of the platinum-sensitive cell line (A2780) drops from 0.7 to 0.1 in 8 hours. In contrast, the NSD value of the platinum-resistant cell line (A2780-CP70) remains nearly constant (0.81 to 0.80) 9 hours after applying drug. The NSD value of normal mouse tissue attached to the tumor xenograft decreases only a little (0.6 to 0.52) compared with A2780. Fig. 2 shows the cisplatin drug response curves. The NSD value of each point is averaged over the entire target. The time between measurements is 24 minutes for A2780-CP70 and normal mouse tissue and is 12 minutes for A2780. The 20 μM cisplatin was applied at time t = 0, and the measurements lasted 9 hours for A2780-CP70 and normal mouse tissue, and lasted 8 hours for A2780. At time=0, the aggressive cell line A2780-CP70 has the highest NSD and the normal mouse mesenterium tissue has lowest NSD (0.6). The NSD of the platinum-sensitive cell line A2780 lies in the middle: 0.7. After applying cisplatin, the NSD curve of A2780 drops immediately. The NSD value of the A2780-CP70 almost doesn't change.
Fig. 2 Motility metric (NSD) of ovarian cancer tumor xenografts responding to 20 μM cisplatin. The x-axis is time (minute), the y-axis is NSD value. The sensitive tumor is A2780, while the insensitive tumor A2780-CP70. Both tumor tissues begin with higher motility than normal mouse tissue. The cisplatin was added at time t=0. The NSD of A2780 dropped very fast and after 8 hours it dropped to 0.1. The NSD of the insensitive tumor A2780-CP70 didn't change during 9 hour peroid. The NSD of normal mouse tissue dropped a little compared with the A2780.
In a further study in a veterinary clinical setting, MCT has been used to predict patient outcome for canine non-Hodgkin's lymphoma. Canine non-Hodgkin's lymphomas are initially characterized by tumoral infiltration of peripheral lymph nodes. Canine non-Hodgkin's lymphomas are diverse in their clinical aggressiveness and response to chemotherapy. The only current biomarker for chemoresponsiveness is tumor cell immunophenotype (i.e. T-cell vs. B-cell origin), but chemoresponsiveness varies tremendously within immunophenotype, which reduces the clinical utility of this biomarker. In our study, we used MCT to measure the heterogeneous response of canine lymphoma biopsies to the standard-of-care doxorubicin. The biodynamic signatures of doxorubicin responsivity ex vivo were correlated with canine patient outcome. These studies have demonstrated, for the first time, the utility of label-free intracellular biodynamic markers to predict therapeutic efficacy for cancer treatment in dogs.
SPECIFIC AIMS The primary study objective is to examine the feasibility of using MCT as a chemosensitivity assay among breast cancer patients receiving neoadjuvant chemotherapy by comparing MCT patterns consistent with chemotherapy response or resistance ex-vivo to confirmed response or resistance to chemotherapy as measured by Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 criteria.
PRIMARY ENDPOINT: Objective pathological response measured at the time of surgery.
