The current paradigm for chemotherapy in the treatment of colorectal cancer (CRC) is based on the maximum-tolerated dose approach (MTD), which uses high doses of cytotoxic agents with the aim of killing tumor cells. However, the toxicity of MTD chemotherapy requires drug delivery in a cyclic manner with rest periods that allow the tumor to recover. In contrast, metronomic chemotherapy (MET) uses low but continuous doses that target the endothelial cells of tumors, thereby directly affecting the development of new blood vessels (angiogenesis). Because the potential impact of MET when used as neoadjuvant chemotherapy (NAC) in CRC has not been explored and contrasted with the effects in perfusion of the standard MTD regimen, this study uses an azoxymethane-induced primary model of CRC to assess changes in perfusion and angiogenic markers. Diffuse reflectance spectroscopy (DRS) was used to longitudinally quantify oxygen saturation (StO2) and compare these changes with the change in expression of VEGF-A between MET and MTD treatments. In the MTD group, an increase in StO2 from 65 to 81% was observed by week 4 of treatment and a reduction of VEGF-A expression from 81% to 16% was observed between weeks 4 and 6.
Ulcerative Colitis (UC) is an idiopathic autoimmune inflammatory disorder of the colon mucosa characterized by leukocyte infiltration into the submucosa and ulcer formation in the epithelium, followed by epithelial cell proliferation and restitution of the mucosal barrier. Optical methods sensitive to tissue oxygen demand and epithelial cell metabolism are ideally suited for clinical guidance of disease progression and remission in UC. Here we present endoscopic reporters of submucosal oxygen demand (i.e. inflammatory-related changes) and epithelial cell metabolism (i.e. mucosal re-epithelialization) within the ulcer bed and surrounding microenvironment in a murine model of UC as putative biomarkers of mucosal healing.
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.
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