KEYWORDS: Tumors, Data modeling, Diffuse reflectance spectroscopy, Reflectivity, Error control coding, In vivo imaging, Tissues, Magnesium, Endoscopy, Data acquisition
Significance: Many studies in colorectal cancer (CRC) use murine ectopic tumor models to determine response to treatment. However, these models do not replicate the tumor microenvironment of CRC. Physiological information of treatment response derived via diffuse reflectance spectroscopy (DRS) from murine primary CRC tumors provide a better understanding for the development of new drugs and dosing strategies in CRC.
Aim: Tumor response to chemotherapy in a primary CRC model was quantified via DRS to extract total hemoglobin content (tHb), oxygen saturation (StO2), oxyhemoglobin, and deoxyhemoglobin in tissue.
Approach: A multimodal DRS and imaging probe (0.78 mm outside diameter) was designed and validated to acquire diffuse spectra longitudinally—via endoscopic guidance—in developing colon tumors under 5-fluoruracil (5-FU) maximum-tolerated (MTD) and metronomic regimens. A filtering algorithm was developed to compensate for positional uncertainty in DRS measurements
Results: A maximum increase in StO2 was observed in both MTD and metronomic chemotherapy-treated murine primary CRC tumors at week 4 of neoadjuvant chemotherapy, with 21 ± 6 % and 17 ± 6 % fold changes, respectively. No significant changes were observed in tHb.
Conclusion: Our study demonstrates the feasibility of DRS to quantify response to treatment in primary CRC models.
Immunotherapy, an emerging field in cancer therapeutics, in colon cancer aims to reduce pre-surgical tumor burden by regulating host immune checkpoints, and when used in combination with neoadjuvant chemotherapy, may improve tumor therapeutic response. One such immune checkpoint is CCL2 (monocyte chemoattractant protein-1)-mediated recruitment of monocytes, which differentiate into tumor-associated macrophages (TAMs) in the tumor microenvironment that promote angiogenesis and tumorigenesis. Thus, CCL2 blockade may play an anti-tumor role via effects on tumor perfusion. However, there have been no studies investigating CCL2 blockade immunotherapy combined with chemotherapy in an animal model of colon cancer. Furthermore, there is a need to longitudinally assess tumor therapeutic response throughout treatment. In this study, CT26 murine colon carcinoma was injected into the flanks of Balb/c mice (n=80) to form tumor allografts. Mice in the key experimental group received combined chemotherapy (5-flurouracil) and immunotherapy (anti-CCL2), with appropriate controls. Tumor therapeutic response was monitored using diffuse reflectance spectroscopy (DRS) by measuring the tumor perfusion metrics, hemoglobin concentration and oxygenation. End-point immunohistochemical analysis was used to quantify TAM fraction (CD68 and DAPI), TAM polarization (iNOS and CD206), and hypoxia (pimonidazole) to spatially and temporally correlate to DRS results. The central hypothesis was that decreasing TAMs via CCL2 blockade alters tumor perfusion, thereby increasing tumor response to 5-fluorouracil. This study may potentially demonstrate an effective immunotherapy approach (CCL2 blockade) and a viable method to longitudinally and non-invasively assess tumor therapeutic response to such immunotherapy (DRS) in mouse allograft models of colon cancer.
Colorectal cancer (CRC) ranks fourth in terms of newly diagnosed cases in the United States (135,430 in 2017); patients with locally advanced disease (Stage II and III) receive 5-fluorouracil (5-FU) and external beam radiotherapy-based neoadjuvant therapy (NAT) prior to surgical resection. However, there are no clinically accepted methods to assess in vivo therapeutic response to NAT.
Optical methods based on diffuse reflectance spectroscopy (DRS) have shown significant promise in predicting response to NAT in breast cancer, but the anatomy of the distal colon requires the use of endoscopically-deployable methods. We have developed a small-diameter (0.78 mm) multimodal optical imaging and diffuse reflectance spectroscopy (DRS) probe which can be deployed via the biopsy port of a commercial veterinary colonoscope (Karl Storz COLOView) to be used in a chemically-induced (azoxymethane (AOM)) orthotopic model.
Total diffuse reflectance measured by the probe was correlated with the reduced scattering (μ’s(λ)) and absorption coefficients (μa(λ)) for λ = 450 – 800nm via a look-up table (LUT). Liquid phantoms were used to create the LUT and validate the measured μ’s and μa values. The LUT has a maximum total reflectance of 0.14 and ranges for μa and μ’s are 0-10 cm-1and 3-18 cm-1, respectively. Error for μ’s and μa has been 10.7±8.8% and 7.9±5.3%, respectively. For the imaging component, circular active area diameter is 325 μm and center-to-center fiber spacing of 3.3 μm.
Building on previous work this DRS approach enables quantification of total hemoglobin (Hb) content, oxygen saturation (SaO2), estimates mean vessel diameter and scattering component, and allows for co-registered highresolution image data of superficial mucosa in vivo of tumor perfusion and microstructure, which can translate to the clinic to help physicians determine the response of tumors to therapy.
Diffuse reflectance spectroscopy (DRS) is a probe-based spectral biopsy technique used in cancer studies to quantify tissue reduced scattering (μs′) and absorption (μa) coefficients and vary in source–detector separation (SDS) to fine-tune sampling depth. In subcutaneous murine tumor allografts or xenografts, a key design requirement is ensuring that the source light interrogates past the skin layer into the tumor without significantly sacrificing signal-to-noise ratio (target of ≥15 dB). To resolve this requirement, a DRS probe was designed with four SDSs (0.75, 2.00, 3.00, and 4.00 mm) to interrogate increasing tissue volumes between 450 and 900 nm. The goal was to quantify percent errors in extracting μa and μs′, and to quantify sampling depth into subcutaneous Balb/c-CT26 colon tumor allografts. Using an optical phantom-based experimental method, lookup-tables were constructed relating μa,μs′, diffuse reflectance, and sampling depth. Percent errors were <10 % and 5% for extracting μa and μs′, respectively, for all SDSs. Sampling depth reached up to 1.6 mm at the first Q-band of hemoglobin at 542 nm, the key spectral region for quantifying tissue oxyhemoglobin concentration. This work shows that the DRS probe can accurately extract optical properties and the resultant physiological parameters such as total hemoglobin concentration and tissue oxygen saturation, from sufficient depth within subcutaneous Balb/c-CT26 colon tumor allografts. Methods described here can be generalized for other murine tumor models. Future work will explore the feasibility of the DRS in quantifying volumetric tumor perfusion in response to anticancer therapies.
Spatial frequency domain imaging (SFDI) is a widefield, noncontact, and label-free imaging modality that is currently being explored as a new tool for longitudinal tracking of cancer therapies in the preclinical setting. We describe a two-layer look-up-table (LUT) inversion algorithm for SFDI that better accounts for the skin (top layer) and tumor (bottom layer) tissue geometry in subcutaneous tumor models. Monte Carlo (MC) simulations were conducted natively in the spatial frequency domain, avoiding discretization errors associated with Fourier or Hankel transforms of conventional MC simulation results. The two-layer LUT was validated using two-layer tissue mimicking optical phantoms, in which the optical property extractions of the bottom (tumor) layer were determined to be within 20% and 11% of the true values for μa and μs′, respectively. A sensitivity analysis was conducted to evaluate how imperfect top layer estimates affect bottom-layer optical property extractions. Finally, the two-layer LUT was used to reanalyze a prior longitudinal data set, which revealed larger therapy-induced changes in optical scattering and a more hypoxic tumor environment compared to the homogeneous LUT. The two-layer LUT described here improves the accuracy of subcutaneous tumor imaging, and the general methodology can be applied for arbitrary multilayer SFDI applications.
Spatial Frequency Domain Imaging (SFDI) is a Diffuse Optical Imaging (DOI) technique that is well suited for preclinical functional imaging. Recently, we have shown that SFDI can successfully be used for longitudinal monitoring of a prostate subcutaneous tumor xenograft, where we have applied a look-up-table (LUT) based approach to extract tissue absorption (μa) and scattering properties (μs’). This LUT assumes a semi-infinite homogeneous medium and simulates reflectance (Rd) in spatial domain, and scales Rd for all μa and μs’ of interest from a single Monte Carlo simulation. However, converting Rd to spatial frequency domain (SFD) and scaling for μs’ may introduces unacceptable errors. Most importantly, the homogeneous model fails to mimic the actual physiology of a subcutaneous tumor, which can be described as a two-layer medium with a thin skin layer above the tumor layer. To overcome these limitations, we have developed a Monte Carlo based two-layer LUT with a wide range of tumor (bottom) layer optical properties, and fixed skin (top) properties. The two-layer LUT will be validated by two-layer silicone phantoms and tested for sensitivity to inaccurate layer assumptions. Additionally, the homogeneous and two-layer LUTs will be used on a large mouse tumor database (n=54 mice monitored over 3 months) to identify how the two-layer LUT can improve accuracy of SFDI by more accurately reflecting in vivo physiology, and reducing discretization and scaling errors. Improved SFDI findings in small animals, in the long run, will help establish clinical DOI tools for early detection of chemotherapy efficacy during treatment.
Locally advanced adenocarcinomas located in the distal rectum are commonly treated via 5-fluorouracil (5-FU)-based neoadjuvant chemoradiation therapy (CRT). The occurrence of pre-operative pathological complete response, or the absence of any histological evidence of residual cancer, is seen in 15-27% of rectal cancer cases. Response to chemotherapeutic agents varies between patients, introducing the need for a system to predict optimal drug combinations. We propose a method of utilizing optical metabolic imaging of in vitro, primary tumor-derived, three-dimensional organoid culture to create specific drug sensitivity profiles, and to rapidly assess a patient’s potential response to drugs. Murine xenografts were developed in Swiss athymic nude mice, using human colorectal adenocarcinoma cell lines, implanted in the flank (RKO, ATCC). Tumors were excised upon reaching a volume of 500mm3 and processed for organoid culture. Organoids were subjected to longitudinal metabolic imaging of metabolic cofactors FAD and NADH for seven days. The resulting images were used to yield an optical redox value on a cell-by-cell basis, determined by the fluorescence intensity ratio of FAD/(FAD+NADH). This data infers proliferative index of the organoids. Beginning on day three, a control vehicle dimethyl sulfoxide, or the cytotoxic agent 5-FU, was added to the organoid growth media in wells, with metabolic imaging performed the same as previously stated. The optical redox values decreased due to the addition of 5-FU, which targets rapidly dividing cells and induces apoptosis. The changes in the optical redox histograms were correlated to markers of cell proliferation (Ki-67) and apoptosis (cleaved caspase-3).
Spatially–resolved diffuse reflectance (SRDR) measurements provide photon path information, and enable layered tissue analysis. This paper presents experimental SRDR measurements on two-layer PDMS skin tissue-mimicking phantoms of varying top layer thicknesses, and bulk phantoms of varying optical properties using concentric multi-pixel photodiode array (CMPA) probes, and corresponding forward Monte Carlo simulations. The CMPA is the most densely packed semiconductor SRDR probe reported to date. Signal contrasts between the single layer phantom and bi-layer phantoms with varying top layer thicknesses are as high as 80%. The mean error between the Monte Carlo simulations and the experiment is less than 6.2 %.
Two-photon excitation of label-free tissue is of increasing interest, as advances have been made in endoscopic clinical application of multiphoton microscopy, such as second harmonic generation (SHG) scanning endoscopy used to monitor cervical collagen in mice1. We used C57BL mice as a model to investigate the progression of gastrointestinal structures, specifically glandular area and circularity. We used multiphoton microscopy to image ex-vivo label-free murine colon, focusing on the collagen structure changes over time, in mice ranging from 10 to 20 weeks of age. Series of images were acquired within the colonic and intestinal tissue at depth intervals of 20 microns from muscularis to the epithelium, up to a maximum depth of 180 microns.
The imaging system comprised a two-photon laser tuned to 800nm wavelength excitation, and the SHG emission was filtered with a 400/40 bandpass filter before reaching the photomultiplier tube. Images were acquired at 15 frames per second, for 200 to 300 cumulative frames, with a field of view of 261um by 261um, and 40mW at sample. Image series were compared to histopathology H&E slides taken from adjacent locations. Quantitative metrics for determining differences between murine glandular structures were applied, specifically glandular area and circularity.
In the non-keratinized epithelia, dysplasia typically arises near the basement membrane and proliferates into the upper epithelial layers over time. We present a non-invasive, multimodal technique combining high-resolution fluorescence imaging and broadband sub-diffuse reflectance spectroscopy (sDRS) to monitor health at various tissue layers. This manuscript focuses on characterization of the sDRS modality, which contains two source-detector separations (SDSs) of 374 μm and 730 μm, so that it can be used to extract in vivo optical parameters from human oral mucosa at two tissue thicknesses. First, we present empirical lookup tables (LUTs) describing the relationship between reduced scattering (μs') and absorption coefficients (μa) and absolute reflectance. LUTS were shown to extract μs' and μa with accuracies of approximately 4% and 8%, respectively. We then present LUTs describing the relationship between μs', μa and sampling depth. Sampling depths range between 210-480 and 260-620 μm for the 374 and 730 μm SDSs, respectively. We then demonstrate the ability to extract in vivo μs', μa, hemoglobin concentration, bulk tissue oxygen saturation, scattering exponent, and sampling depth from the inner lip of thirteen healthy volunteers to elucidate the differences in the extracted optical parameters from each SDS (374 and 730 μm) within non-keratinized squamous epithelia.
Many cases of epithelial cancer originate in basal layers of tissue and are initially undetected by conventional microendoscopy techniques. We present a bench-top, fiber-bundle microendoscope capable of providing high resolution images of surface cell morphology. Additionally, the microendoscope has the capability to interrogate deeper into material by using diffuse reflectance and broadband diffuse reflectance spectroscopy. The purpose of this multimodal technique was to overcome the limitation of microendoscopy techniques that are limited to only visualizing morphology at the tissue or cellular level. Using a custom fiber optic probe, high resolution surface images were acquired using topical proflavine to fluorescently stain non-keratinized epithelia. A 635 nm laser coupled to a 200 μm multimode fiber delivers light to the sample and the diffuse reflectance signal was captured by a 1 mm image guide fiber. Finally, a tungsten-halogen lamp coupled to a 200 μm multimode fiber delivers broadband light to the sample to acquire spectra at source-detector separations of 374, 729, and 1051 μm. To test the instrumentation, a high resolution proflavine-induced fluorescent image of resected healthy mouse colon was acquired. Additionally, five monolayer poly(dimethylsiloxane)-based optical phantoms with varying absorption and scattering properties were created to acquire diffuse reflectance profiles and broadband spectra.
Optical phantoms are used in the development of various imaging systems. For certain applications, the development of thin phantoms that simulate the physical size and optical properties of tissue is important. Here, we demonstrate a method for producing thin phantom layers with tunable optical properties using poly(dimethylsiloxane) (PDMS) as a substrate material. The thickness of each layer (between 115 and 880 μm) was controlled using a spin coater. The reduced scattering and absorption coefficients were controlled using titanium dioxide and alcohol–soluble nigrosin, respectively. These optical coefficients were quantified at six discrete wavelengths (591, 631, 659, 691, 731, and 851 nm) at varying concentrations of titanium dioxide and nigrosin using spatial frequency domain imaging. From the presented data, we provide lookup tables to determine the appropriate concentrations of scattering and absorbing agents to be used in the design of PDMS-based phantoms with specific optical coefficients. In addition, heterogeneous phantoms mimicking the layered features of certain tissue types may be fabricated from multiple stacked layers, each with custom optical properties. These thin, tunable PDMS optical phantoms can simulate many tissue types and have broad imaging calibration applications in endoscopy, diffuse optical spectroscopic imaging, and optical coherence tomography, etc.
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