Macrophage-targeted anti-CCL2 immunotherapy enhances tumor sensitivity to 5-fluorouracil in a Balb/c-CT26 murine colon carcinoma model measured using diffuse reflectance spectroscopy.

BACKGROUND: Immunotherapy in colorectal cancer (CRC) regulates specific immune checkpoints and, when used in combination with chemotherapy, can improve patient prognosis. One specific immune checkpoint is the recruitment of circulating monocytes that differentiate into tumor-associated macrophages (TAMs) and promote tumor angiogenesis. Changes in vascularization can be non-invasively assessed via diffuse reflectance spectroscopy using hemoglobin concentrations and oxygenation in a localized tumor volume. In this study, we examine whether blockade of monocyte recruitment via CCL2 (macrophage chemoattractant protein-1) leads to enhanced sensitivity of 5-fluorouracil (5-FU) in a CT26-Balb/c mouse model of CRC. It was hypothesized that the blockade of TAMs will alter tumor perfusion, increasing chemotherapy response. A subcutaneous tumor model using Balb/c mice injected with CT26 colon carcinoma cells received either a saline or isotype control, anti-CCL2, 5-FU, or a combination of anti-CCL2 and 5-FU. RESULTS: Findings show that 12 days post-treatment, monocyte recruitment was significantly reduced by approximately 61% in the combination group. This shows that the addition of anti-CCL2 to 5-FU slowed the fold-change (change from the original measurement to the final measurement) in tumor volume from Day 0 to Day 12 (~ 5 fold). Modest improvements in oxygen saturation (~ 30%) were observed in the combination group. CONCLUSION: The findings in this work suggest that the blockade of CCL2 is sufficient in the reduction of TAMs that are recruited into the tumor microenvironment and has the ability to modestly alter tumor perfusion during early-tumor response to treatment even though the overall benefit is relatively modest.

Diffuse reflectance spectroscopy to monitor murine colorectal tumor progression and therapeutic response (Erratum).

The erratum notes a correction to Fig. 5 for the published article.

Diffuse reflectance spectroscopy to monitor murine colorectal tumor progression and therapeutic response.

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.

Efficacy and clinical monitoring strategies for immune checkpoint inhibitors and targeted cytokine immunotherapy for locally advanced and metastatic colorectal cancer.

Colorectal cancer (CRC) is the fourth most common cancer type and is the second leading cause of cancer deaths annually in the United States. Conventional treatment options include postoperative (adjuvant) and preoperative (neoadjuvant) chemotherapy and radiotherapy. Although these treatment modalities have shown to decrease tumor burden, a major limitation to chemothearpy/radiotherapy is the high recurrence rate in patients. Immune-modulation strategies have emerged as a promising new therapeutic avenue to reduce this recurrence rate while minimizing undesirable systemic side effects. This review will focus specifically on the mechanisms of monoclonal antibodies: immune checkpoint inhibitors and cytokines, as well as current drugs approved by the Food and Drug Administration (FDA) and new clinical/pre-clinical trials. Finally, this review will investigate emerging methods used to monitor tumor response post-treatment.

Effects of isoflurane anesthesia on physiological parameters in murine subcutaneous tumor allografts measured via diffuse reflectance spectroscopy.

Diffuse reflectance spectroscopy (DRS) has been used in murine studies to quantify tumor perfusion and therapeutic response. These studies frequently use inhaled isoflurane anesthesia, which depresses the respiration rate and results in the desaturation of arterial oxygen saturation, potentially affecting tissue physiological parameters. However, there have been no controlled studies quantifying the effect of isoflurane anesthesia on DRS-derived physiological parameters of murine tissue. The goal of this study was to perform DRS on Balb/c mouse (n = 10) tissue under various anesthesia conditions to quantify effects on tissue physiological parameters, including total hemoglobin concentration, tissue oxygen saturation, oxyhemoglobin and reduced scattering coefficient. Two independent variables were manipulated including metabolic gas type (pure oxygen vs. medical air) and isoflurane concentration (1.5 to 4.0%). The 1.5% isoflurane and 1 L/min oxygen condition most closely mimicked a no-anesthesia condition with oxyhemoglobin concentration within 89% ± 19% of control. The time-dependent effects of isoflurane anesthesia were tested, revealing that anesthetic induction with 4.0% isoflurane can affect DRS-derived physiological parameters up to 20 minutes post-induction. Finally, spectroscopy with and without isoflurane anesthesia was compared for colon tumor Balb/c-CT26 allografts (n = 5) as a representative model of subcutaneous murine tumor allografts. Overall, isoflurane anesthesia yielded experimentally-induced depressed oxyhemoglobin, and this depression was both concentration and time dependent. Investigators should understand the dynamic effects of isoflurane on tissue physiological parameters measured by DRS. These results may guide investigators in eliminating, limiting, or managing anesthesia-induced physiological changes in DRS studies in mouse models.

Sampling depth of a diffuse reflectance spectroscopy probe for in-vivo physiological quantification of murine subcutaneous tumor allografts.

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.

Two-layer inverse model for improved longitudinal preclinical tumor imaging in the spatial frequency domain.

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.

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis.

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of spectroscopy techniques. Combining imaging and spectroscopy techniques into a single optical probe may provide a more complete analysis of tissue health. In this article, two dissimilar modalities are combined, high-resolution fluorescence microendoscopy imaging and diffuse reflectance spectroscopy, into a single optical probe. High-resolution fluorescence microendoscopy imaging is a technique used to visualize apical tissue micro-architecture and, although mostly a qualitative technique, has demonstrated effective real-time differentiation between neoplastic and non-neoplastic tissue. Diffuse reflectance spectroscopy is a technique which can extract tissue physiological parameters including local hemoglobin concentration, melanin concentration, and oxygen saturation. This article describes the specifications required to construct the fiber-optic probe, how to build the instrumentation, and then demonstrates the technique on in vivo human skin. This work revealed that tissue micro-architecture, specifically apical skin keratinocytes, can be co-registered with its associated physiological parameters. The instrumentation and fiber-bundle probe presented here can be optimized as either a handheld or endoscopically-compatible device for use in a variety of organ systems. Additional clinical research is needed to test the viability of this technique for different epithelial disease states.

Quantitative structural markers of colorectal dysplasia in a cross sectional study of ex vivo murine tissue using label-free multiphoton microscopy.

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 vivo measurement of non-keratinized squamous epithelium using a spectroscopic microendoscope with multiple source-detector separations.

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.

Towards monitoring dysplastic progression in the oral cavity using a hybrid fiber-bundle imaging and spectroscopy probe.

Intraepithelial dysplasia of the oral mucosa typically originates in the proliferative cell layer at the basement membrane and extends to the upper epithelial layers as the disease progresses. Detection of malignancies typically occurs upon visual inspection by non-specialists at a late-stage. In this manuscript, we validate a quantitative hybrid imaging and spectroscopy microendoscope to monitor dysplastic progression within the oral cavity microenvironment in a phantom and pre-clinical study. We use an empirical model to quantify optical properties and sampling depth from sub-diffuse reflectance spectra (450-750 nm) at two source-detector separations (374 and 730 µm). Average errors in recovering reduced scattering (5-26 cm(-1)) and absorption coefficients (0-10 cm(-1)) in hemoglobin-based phantoms were approximately 2% and 6%, respectively. Next, a 300 µm-thick phantom tumor model was used to validate the probe's ability to monitor progression of a proliferating optical heterogeneity. Finally, the technique was demonstrated on 13 healthy volunteers and volume-averaged optical coefficients, scattering exponent, hemoglobin concentration, oxygen saturation, and sampling depth are presented alongside a high-resolution microendoscopy image of oral mucosa from one volunteer. This multimodal microendoscopy approach encompasses both structural and spectroscopic reporters of perfusion within the tissue microenvironment and can potentially be used to monitor tumor response to therapy.

Fiber-bundle microendoscopy with sub-diffuse reflectance spectroscopy and intensity mapping for multimodal optical biopsy of stratified epithelium.

Early detection of structural or functional changes in dysplastic epithelia may be crucial for improving long-term patient care. Recent work has explored myriad non-invasive or minimally invasive "optical biopsy" techniques for diagnosing early dysplasia, such as high-resolution microendoscopy, a method to resolve sub-cellular features of apical epithelia, as well as broadband sub-diffuse reflectance spectroscopy, a method that evaluates bulk health of a small volume of tissue. We present a multimodal fiber-based microendoscopy technique that combines high-resolution microendoscopy, broadband (450-750 nm) sub-diffuse reflectance spectroscopy (sDRS) at two discrete source-detector separations (374 and 730 µm), and sub-diffuse reflectance intensity mapping (sDRIM) using a 635 nm laser. Spatial resolution, magnification, field-of-view, and sampling frequency were determined. Additionally, the ability of the sDRS modality to extract optical properties over a range of depths is reported. Following this, proof-of-concept experiments were performed on tissue-simulating phantoms made with poly(dimethysiloxane) as a substrate material with cultured MDA-MB-468 cells. Then, all modalities were demonstrated on a human melanocytic nevus from a healthy volunteer and on resected colonic tissue from a murine model. Qualitative in vivo image data is correlated with reduced scattering and absorption coefficients.

Design and validation of a diffuse reflectance and spectroscopic microendoscope with poly(dimethylsioxane)-based phantoms.

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.

Characterization of thin poly(dimethylsiloxane)-based tissue-simulating phantoms with tunable reduced scattering and absorption coefficients at visible and near-infrared wavelengths.

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.