Drug screening for pharmaceutical development has relied on two-dimensional (2-D) cell culture because of the ease of imaging thin layers of cells using conventional as well as advanced forms of microscopy. However, the 2-D environment is not representative of biological tissue, as cells live in a three-dimensional (3-D) microenvironment with physical as well as chemical contacts to their local environment. 3-D cell culture systems overcome many of the limitations of traditional 2-D monolayer cell culture systems by mimicking more closely the complex cellular heterogeneity and tumor microenvironmental conditions.1 In addition, it is recognized that cellular dimensionality affects cancer drug sensitivity or resistance in 3-D cultures relative to 2-D monolayers.2 Consequently, there is a growing trend toward 3-D culture to capture more biologically relevant tissue response to applied compounds.3–9 However, deep-tissue optical imaging techniques capable of measuring pharmaceutical effects inside living tissue are currently lacking. Candidate imaging approaches include confocal and nonlinear microscopy,10 light-sheet microscopy,11 and optical coherence tomography (OCT).12,13 Of these, only OCT has significant depth penetration (millimeters) to move deeper than surface layers of cells. OCT has been used to image pharmaceutical responses of 3-D tissues,14–17 but it lacks high-content screening capabilities that are essential to characterize complex pharmaceutical response of tissue.
Biodynamic imaging (BDI)18–20 is an optical imaging technology related to en face OCT,21 but with enhanced partially coherent speckle generated by broad-area illumination with coherence detection through digital holography.22–24 BDI penetrates up to 1 mm into living tissue and returns high-content information in the form of dynamic light scattering across a broad spectral range.25,26 The fluctuation frequencies relate to Doppler frequency shifts caused by light scattering from the subcellular constituents that are in motion. The speeds of intracellular dynamics range across four orders of magnitude from tens of nanometers per second (cell membrane)27–30 to tens of microns per second (organelles, vesicles).31–34 In the near-infrared backscattering geometry, these speeds correspond to Doppler frequencies from 0.005 to 50 Hz.
Frequency-domain decomposition of the light fluctuations using tissue dynamics spectroscopy19 produces broad-band fluctuation spectra that encode the wide variety of subcellular motions. When pharmaceutical compounds are applied to the tissue, dynamic cellular processes are modified, and these modifications appear as changes in the fluctuation spectra. By applying reference compounds with known mechanisms of action, a library of drug-response spectrograms can be generated against which drug screens may be compared, gaining information about the effect of the compound on cellular processes such as necrosis and apoptosis.35 This type of phenomenological assay is known as a phenotypic profile. Phenotypic profiling has seen a resurgence in recent years as a more systems-based approach to drug discovery and development36 that captures the complex interplay of cellular processes affected by the drug candidate. Phenotypic profiling of dynamics using digital holography on 2-D culture has been performed,37 but does not probe the 3-D microenvironment of tissues.
This paper describes the application of BDI for the phenotypic profiling of responses to chemotherapy using distinct 3-D tissue culture approaches. Our results demonstrate that different culture approaches, even when applied to the same cancer cell line, produce tissues with distinct cellular sensing of chemotherapy, as measured by BDI.
Materials and Methods
Several methods are used to generate 3-D multicellular tumor spheroids including: (i) Bioreactors(BRs) in which cells or spheroids aggregate in the chamber;38–40 (ii) nonadherent plates that prevent attachment;41,42 (iii) hanging-drop (HD) plates where cells are grown in a suspended droplet to form one single spheroid;43–46 (iv) multicellular layers;47–49 and (v) organoid culture50,51 among others. In this paper, BDI was used to assess the different cellular responses to paclitaxel of cancer spheroids obtained by using the three most common approaches: BR, HD, and nonadherent plate growth. Within these growth approaches, there are important differences that may relate to the downstream success rate for drug screening and development.
Three-Dimensional Tissue Culture
Three different multicellular spheroid tissue growth methods were used for 3-D tissue culture: (1) a Synthecon (Houston, Texas) rotating BR, (2) Corning (Corning, New York) U-bottom (UB) spheroid plates, and (3) 3D Biomatrix (Ann Arbor, Michigan) HD plates. Two cell lines, A2780 (human ovarian cancer) and DLD-1 (human colon adenocarcinoma), were obtained from the American Type Culture Collection (Manassas, Virginia) and grown at 37°C in a humidified 5% incubator in RPMI-1640 medium supplemented with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, Georgia), of penicillin, and of streptomycin (Sigma). To grow spheroids in the BR, the cells from a flask () were placed in a 50-mL capacity rotating drum BR in complete growth medium. The medium was refreshed (30%) every other day until the cells formed spheroids of optimal experimental size (300 to in diameter). Cells were seeded at a concentration of 5000/well in Corning spheroid plates and 5000/drop in the 3D Biomatrix HD plate. Spheroids formed within 24 to 48 h in these plate-based methods.
Ex Vivo Tumor Explants
Established cancer cell lines and xenografts have been the mainstay of cancer research for the past several decades. To date, the analysis of drug sensitivity and resistance in both established cancer cell lines and xenografts has been indispensable. However, while testing of compounds in established cancer cell lines is often not predictive of clinical efficacy, corresponding in vivo studies in animals are very expensive and time consuming. Therefore, heightened attention is being paid toward ex vivo analysis of tumor explants due to the greater understanding that cancer therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Ex vivo treatment response analysis could become a standard tool in preclinical and clinical development of cancer therapeutics and is envisioned as a step toward a personalized medicine approach for new therapeutic development strategies.52,53
Tumor xenografts were generated in nude mice using human ovarian carcinoma cell lines (A2780, SKOV-3). Animal procedures were approved by the Indiana University School of Medicine Animal Care and Use Committee and were in accordance with federal regulations. Female nude mice (nu/nu BALB/c) 7- to 8-weeks old (Harlan, Indianapolis, IN) were subcutaneously injected in the right flank with cells or orthotopically injected in one of the ovaries. The mice were euthanized 2 to 4 weeks after the injection and the tumor xenografts removed. Tumor xenografts were diced into pieces and immobilized in 96-well cell culture plates in a thin layer of 1% low gel-temperature agarose made up in HEPES buffered RPMI 1640 medium (ATCC, Manassas, Virginia).
BDI was performed with an optical instrument that captures backscattered light from biological samples and records digital holograms on a CCD Fourier-plane chip. The light source is a continuous-wave low-coherence light source (Superlum, Cork, Ireland) with a 20-mW output intensity at a wavelength of 850 nm and a bandwidth of 15 nm with a coherence length of . The optical section thickness is significantly broadened by multiple scattering with increasing broadening with increasing gated depth. The BDI system is a Mach–Zehnder interferometer shown in Fig. 1. The light path is divided into a signal and reference arm by a polarizing beam splitter with a variable polarizer to adjust the relative intensities in the signal and reference arms. Dynamic light scattering is performed in a back-scatter geometry because the sensitivity to Doppler frequency shifts depends on the momentum transfer vector that selects longitudinal motion along the backscatter direction. The maximum backscattering momentum transfer is given by , where is the refractive index of the tissue and is the free-space wavelength. The reduced wavelength inside the medium is given by . Assuming a refractive index and a free-space wavelength of 840 nm, the reduced wavelength is equal to 50 nm. The light scattered by the living biological sample is collected by a long focal-length lens and transformed to a Fourier plane (FP) where the CCD pixel array is placed. The reference wave is incident on the CCD array at a small angle of 3 deg relative to the signal axis, creating an off-axis digital hologram with a fringe spacing of on the camera chip. A digital hologram is acquired on the FP of the optical imaging system, and this Fourier-domain hologram is transformed using an FFT algorithm into the image domain. The transformation performs two functions: (1) demodulating the spatial carrier frequency represented by the holographic interference fringes and (2) coherence-gating the low-coherence light to a specified depth inside the tissue sample.
The coherence-gating role of digital holography creates a full-frame OCT section of the tumor spheroid at a fixed depth. The reconstructed section is highly speckled, which normally is considered to be an undesirable side effect in OCT, but dynamic speckle for our application provides the basis for BDI. BDI captures successive frames at a fixed coherence gate at a frame rate of 25 Hz. In dynamic light scattering, there are two limits related to the characteristic fluctuation frequency depending on the mean speeds of intracellular motions. The Doppler limit corresponds to mean free flight distances longer than the reduced wavelength, for , where is the intracellular speed and is the persistence time for the transport. The opposite limit is the diffusion limit when . In this limit, the diffusion is still dominated by random flights rather than thermally driven Brownian motion, but the transport is a random walk, driven by active transport such as molecular motors or cytoskeletal restructuring. The diffusion coefficient in this limit is given by , and the diffusion and Doppler angular frequencies, in the respective limits, are
Living tissue is highly complex, with a wide range of speeds and persistence times. The measured knee frequency (half-width half-maximum of the spectrum) for the fluctuation spectrum scales as54–57 Speeds on the order of are associated with nuclear motions and membrane dynamics.28,33,58 Speeds on the order of and above are associated with organelle transport and cytoskeletal restructuring.59–61
A common metric that characterizes tissue health is the normalized standard deviation (NSD), also known as temporal speckle contrast,62 that is calculated from the standard deviation of the intensity divided by the average intensity. Large fluctuations in the dynamic speckle images result in high NSD values, and a 2-D map of NSD values gives a visual measure of motility, called motility contrast imaging (MCI). The MCI map displays the NSD from a biological sample as a representation of the activity/health of the sample. A high NSD can indicate vitality of the sample, high proliferation, or it may represent a high overall metabolic rate. For example, 3-D spheroids of known aggressive cancer cell lines with short doubling times and high metabolism typically have a higher NSD value than less aggressive cancers for spheroids of equivalent health. For an exponential probability distribution function of intensities, the standard deviation is equal to the mean, and therefore under ideal sampling . However, for finite sampling, the NSD is less than unity and depends on the finite-sampling time as well as on the characteristic persistence times of the subcellular motion. Figure 2 shows the NSD versus the scaling quantity for a set of 100 Monte Carlo simulations using random particle flights with speeds of 0.1 and with persistence times of 0.1 and 1 s. The time is the total acquisition time, and is the persistence time. The NSD approaches 1 as and NSD changes rapidly when . The condition for is . Because and are fixed parameters of the experiment, changes in NSD induced by applied therapies are caused by changes in the product, capturing the influence of the therapy on the physiological motions of the living sample. The common behavior for different velocities and persistence times is described by the relationship
The time traces of the fluctuating intensities are transformed into the frequency domain as a spectral power density denoted by . The measured power spectrum is a function of the frame rate of the acquisition and exposure time of the shutter. In particular, the Nyquist theorem states that the highest frequency that can be resolved is , where fps stands for frames per second. In addition, during the data acquisition, the detection bandwidth is set by the exposure time as , where is the exposure time that “freeze-frames” motion, like a strobe, between the Nyquist frequency and the detection bandwidth. The integral of the spectral power between the Nyquist frequency and the detection bandwidth appears as a baseline at the Nyquist frequency. This is called the Nyquist floor and is not, in general, a noise floor but contains information about fast vesicle motions with frequencies up to . The spectrum also has a noise floor that can contribute to the integral, but this contribution is usually small relative to the Nyquist floor in active tissues.
Biodynamic Growth Characterization
A selection of representative power spectra is shown in Fig. 3 for BR, HD, and UB plate spheroids as well as living biopsies. In Fig. 3(a), the HD spheroids have the highest knee frequency because they are more active than the other two growth methods. Figure 3(b) shows the spectra of a mouse ovarian xenograft biopsy and a human esophageal biopsy compared with tumor spheroids from a BR. The NSD of the tumor biopsies have mean values around 0.8, which are similar to values for the BR sample, and the power spectra have similar knee frequencies around 0.05 Hz. The similarity between the biopsies and the BR tumor spheroids provides support for the conclusion that the BR sample performance is closer to the biopsy than the HD and UB methods. This could reflect the longer culture period in the BR, enabling the formation of extracellular matrix (ECM), and denser cellular adhesions,63 rendering BR-grown spheroids more similar to in vivo grown tumors.
Motility contrast images (MCI) of the A2780 and DLD-1 cell lines under the three growth methods are shown in Fig. 4(a). The NSD increases from the BR to UB to HD for each cell line. An averaged NSD of 0.79 for BR DLD and 0.82 for BR A2780, and a higher NSD of 0.9 for the HD DLD and A2780 spheroids were measured. The BR usually takes several weeks to grow tumor spheroids to the optimal size, which allows it to develop more ECM and complex cellular adhesions than the other methods.64 Conversely, the UB and HD methods take 1 to 2 days to accomplish the growth, and hence they tend to be looser aggregates of cells. It is typical for the BR spheroids to have low motility in the core of the spheroid because the cells in the core area have been exposed to lower oxygen and nutrients for a longer period of time, which can be seen from the MCI of the BR-grown A2780 spheroid in Fig. 4(a). Unlike the BR spheroids, the UB and HD spheroids tend to be more uniform because the cells are less dense, and nutrients can be transported more easily from the outer environment into the spheroids.49,65–67
The average NSD values and knee frequencies are shown in Figs. 4(b) and 4(c). The sample numbers for these data are A2780: BR , UB , and HD ; DLD1: BR , UB , and HD . The BR method has the smallest average NSD and knee frequencies, and the HD method has the highest average NSD values and knee frequencies. On the other hand, the BR samples have large sample-to-sample variability with varying spheroid metabolic and physiological activity. The variation is diverse because of the long duration of the BR growth. In the same batch, some spheroids may be actively growing, while others may be dying out. The HD method produces spheroids with higher NSD and knee frequencies with smaller variability. Figures 5(a) and 5(b) show the relation between NSD versus knee frequency and NSD versus backscattering brightness. The HD and UB growth techniques produce homogeneous spheroids, and the knee frequency and NSD tend to be high. Unlike HD and UB growth techniques, the BR growth technique produces spheroids with a wide variation. In Fig. 5(a), the NSD and knee frequency vary from 0.6 to 0.9, and 0.01 to 0.1, respectively. A similar trend occurs in the backscattering brightness in Fig. 5(b). Spheroids from HD and UB plates have low backscattering brightness. BR spheroids have higher backscattering brightness because the BR spheroids are structurally heterogeneous. Spheroids from HD and UB techniques are more uniform, but have low ECM and low adhesion density. Therefore, HD and UB plates are good for high-viability and high-uniformity requirements in drug screening, whereas the BR growth is more suitable for experiments that are dependent upon more developed ECM and cell junctions.
Biodynamic Drug Response
When a drug is applied to a sample, or the environmental conditions change, the relative power density at different frequencies is altered. This change is captured through the differential relative spectral power density defined as63,68 Paclitaxel prolongs activation of the mitotic checkpoint, triggering apoptosis, or reversion to the G-phase of the cell cycle without cell division. Paclitaxel was added into the growth medium at time at a concentration of . In Fig. 6(a), the spectrograms show the relative spectral density increase/decrease in response to the drug. The frequency axis is logarithmic and extends from 0.01 to 12.5 Hz. The time axis extends from to 12 h, in which the application of the dose is made at . There are 4 h of baseline and 12 h of dose response. The A2780 and DLD-1 spheroids have similar responses to the paclitaxel, with strong enhancement in the low frequency () band and inhibited mid frequency (0.1 to 1 Hz).
The drug-response spectrograms exhibit recognizable features that occur in characteristic frequency ranges at characteristic times after a dose is applied. There are many ways that the time-frequency plane can be partitioned and quantified into a feature vector. For the spectrograms in Fig. 6(a), we quantified three feature metrics called SDIP, QDIP, and APOP. The two features SDIP and QDIP are obtained by multiplying the spectrograms by linear masks that represent red shifted (spectral weight moving to lower frequencies) and mid-enhanced (increased spectral weight in central frequencies, and decreased spectral weight at high and low frequencies) frequency patterns, respectively. The feature APOP is a nonlinear mask that measures combined enhanced low frequencies and enhanced high frequencies that captures organelle transport and blebbing processes during apoptosis.58 The three biodynamic biomarkers are shown in Fig. 6(b) for the two cell lines and three growth methods. The biomarkers are consistent between the A2780 and DLD-1 cell lines for the HD and UB growth. However, the SDIP biomarker for the BR-grown samples have different signs between the two cell lines. Here again the HD and UB techniques show better homogeneity and similarities between these growth techniques, but the BR samples are less consistent. This is an important conclusion for researchers seeking to perform consistent drug screens in 3-D tissue.
Drug sensitivity can depend on adhesion density through the interplay between adhesions and the cytoskeleton that is affected by paclitaxel. Adhesion density also depends on the growth method.60 Two consistent metrics that can be tracked in time are the brightness of the samples and the temporal speckle contrast (NSD). Temporal speckle contrast is a direct measure of overall activity, and backscatter brightness (BB) increases with increasing optical heterogeneity, which parallels cellular structural heterogeneity. The time-dependences of NSD and BB are shown in Fig. 7. The application of paclitaxel decreases the temporal speckle contrast for all cell line and growth cases because the drug stabilizes microtubules and prevents mitosis and inhibits cell shape changes. The sample brightness, on the other hand, shows no consistent trend among the cell lines or growth techniques.
BDI can be used to extract direct pharmacodynamic effects by isolating mitotic events inside tissue. Mitotic events are detected in the mid-Doppler frequency band between 0.1 and 1 Hz. Local pixel-based spectrograms are investigated for signatures in this specific spectral range and are scored positive for a mitotic event when the signature occurs within a resolution voxel. To provide sufficient signal-to-noise, the spectra of four pixels are averaged. Because mitosis occurs at a rate of approximately once per day per cell, and each local 4-pixel volume has about 20 cells, there is mitotic event per hour in each local spectrogram. Figures 8(a) and 8(b) show the normalized mitotic index change after paclitaxel was applied to tumor spheroids from the two cell lines A2780 and DLD-1 under the different growth approaches. Both cell line types show a pronounced decrease after paclitaxel was applied, compared to the control sample. For both types of spheroids, the response time is around 2.5 h. Interestingly, the mitotic index change is almost identical for each cell line and growth condition. The diffusion rate of paclitaxel into these spheroids takes only minutes because paclitaxel is a small molecule with molecular weight . The 2-to-3 h response captures the gradual arrest of cell proliferation as cells move through the cell cycle and are arrested at the mitotic checkpoint. The ability of BDI to measure the mitotic index in living tissue responding to cytostatics represents an improvement over available current methods such as long-term growth-delay studies.
Discussion and Conclusions
BDI can be related to previous work associated with dynamic measurements of cellular processes using coherence-domain techniques that include dynamic light scattering OCT,69 field-based dynamic light scattering microscopy,70 diffusion-sensitive OCT,71 and dynamic full-field OCT.72 BDI uses coherence-domain detection similar to each of these techniques, but with an emphasis on developing a full speckle field at tissue scales that generates a high dynamic range with high signal-to-noise per voxel. This makes it possible to differentiate subtle differences in the action of xenobiotics on tissue activity. In addition, BDI penetrates deeply into dense tissue up to 1-mm deep because imaging aberrations do not degrade its resolution. The tradeoff between resolution and dynamic range has established a compromise at this resolution scale that is also compatible with off-axis holography that might limit higher-resolution approaches. The BDI system used in this work had a Mach–Zender configuration and was not common path (which has the advantage of low sensitivity to mechanical vibrations73), but the system has passive vibration isolation that removes vibration background. In addition, the digital image reconstruction takes the modulus of the Fourier-reconstructed image and does not use the complex values, which makes phase stability unnecessary for this implementation.
The ability to monitor a living sample nondestructively over time opens an avenue for longitudinal drug exposure studies. This paper focused on the use of BDI for the assessment of physiological and pharmacological properties of 3-D tissues grown using different culture methods. Use of 3-D tissue is a new trend in cancer drug screening, but there has been a proliferation of different methods to grow 3-D tissues with a lack of complete characterization of the different drug responses. Because drug screening is already fraught with inconsistencies leading to difficulties moving promising new compound leads into approved drugs, it is critically important to establish the relevant tissue properties that best recapitulate the targeted in vivo behavior. Therefore, this paper focused on the development of BDI as a new tool for assessment of drug response in various types of 3-D tissue, including in-vitro and ex-vivo models.
The four growth techniques considered in this paper differ in time and manner of growth. The nonadherent UB plates have become one of the easiest means to generate tumor spheroids in only a matter of two or three days, and hence has gained popularity, but the rapid growth provides no time for the development of tight tissue structure or the generation of ECM, which can be an important factor in inter- and intracellular signaling. The HD technique is similar to the UB approach, but produces more homogeneous behavior of the samples in terms of their biodynamic signatures. The BR growth is notably different from these other two tissue culture methods, taking up to 1 month to grow a batch of tumor spheroids. The slow growth provides sufficient time for BR-grown spheroids to develop traces of ECM and also tighter tissue structures. The tighter tissue structures present low diffusion coefficients for oxygen and nutrients and hinders the out diffusion of metabolites, creating hypoxic cores for moderate-sized tumor spheroids and even necrotic cores for spheroids with diameters larger than . The BR-grown cultured spheroids are most similar to biopsies in their biodynamic signatures, including the wide heterogeneity of tissue biodynamic properties.
David Nolte and John Turek have a financial interest in Animated Dynamics Inc., which is licensing biodynamic imaging technology from the Office of Technology Commercialization of Purdue University.
This work was supported by grants NSF 1263753-CBET and NIH NIBIB 1RO1EB016582-02. We also thank 3-D Biomatrix for supplying the materials to perform the hanging-drop growth.
Hao Sun received his PhD in physics from Purdue University in 2016. He studied dynamic holography in both semiconductors and biomedical optics under the direction of Dr. David Nolte. He currently works on mobile search ads for cars at Google, Mountain View, California, USA.
Daniel Merrill received his PhD from Purdue University in 2016. He completed his doctoral studies under the direction of Dr. David Nolte with whom he developed biodynamic imaging into a predictive assay for patient therapy selection. He currently teaches physics at Indiana State University where he is developing undergraduate laboratory courses that integrate cross-disciplinary experimental design.
Ran An received his BS degree from the University of Science and Technology, China, in 2008, and his PhD from the Department of Physics and Astronomy at Purdue University in 2014, developing the science and technology of biodynamic imaging. He is a technical founder and the chief technology officer of Animated Dynamics, Inc., a biotech start-up located in Indianapolis that is commercializing biodynamic imaging technology and services.
John Turek received his PhD from the University of Illinois at Urbana in Veterinary Medical Science. He is a professor in the Department of Basic Medical Sciences at Purdue University College of Veterinary Medicine. For the past 16 years, his research has been in the area of biodynamic imaging with a focus on the use of 3-D cell cultures and BDI for derisking drug discovery and for cancer chemotherapy selection.
Daniela Matei received her MD from the University of Medicine and Pharmacy “Carol Davila” and completed postgraduate training at SUNY at Stony Brook and at UCLA. She is a professor in the Department of Obstetrics and Gynecologyoined at Northwestern University Feinberg School of Medicine specializing in ovarian cancer. Her laboratory studies mechanisms of metastasis and therapeutics. She is engaged in clinical research testing therapies for ovarian cancer.
David D. Nolte received his BS degree from Cornell University in 1981 and his PhD from the University of California at Berkeley in 1988, followed by a postdoctoral appointment at AT&T Bell Labs. He is the E.M Purcell distinguished professor of physics at Purdue University. He has been elected as a fellow of the Optical Society of America, a fellow of the American Physical Society, and a fellow of the AAAS. He is the technical founder of two biotech startup companies.