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

Abstract

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.

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

“…9(a)], in contrast with values extracted from subcutaneous models, where values below 50% have been reported. 23,57 We hypothesize that the observed difference can be partially explained by the size of the malignancy, as primary CRC tumors are much smaller (diameter ∼1 mm) than their subcutaneous counterparts, which accordingly would be more hypoxic due to changes in perfusion and necrosis as a result to tumor growth. 58 But it is also possible that higher StO 2 values are caused by the blood present on the readily visible superficial vasculature in all groups [ Fig.…”

“…31,32 The phantom optical properties, system configuration, and LUT appear in Figs Sampling depth of the DRS configuration of the multimodal probe was defined as the depth reached by 50% of the photons and calculated as reported elsewhere. 23,33 Methods are described in detail in Sampling Depth in the Supplementary Material.…”

“…The filtered in-vivo data were used to quantify optical properties and physiological values from tissue (StO 2 , tHb, HbO 2 , and Hb) using the inverse LUT model between 475 and 685 nm using constraining equations and boundary conditions as reported elsewhere. 23,28 Pigment packaging correction was not introduced in the absorption coefficient as the analysis was performed >400 nm. 29 Detailed methodology is presented in DRS post-processing (Inverse LUT model) in the Supplementary Material.…”

“…Our group has previously used DRS in subcutaneous models of CRC, 23 demonstrating the feasibility and advantages of extracting in-vivo physiological information. However, as the diameter of a mouse colon is below 4 mm and the thickness of the colonic wall ranges between 180 and 800 μm, 6,7 the miniaturization of DRS instrumentation presents different challenges: the probe needs to be miniaturized while maintaining an adequate signal-to-noise ratio, 24 and peristalsis and subject breathing can both cause displacement between the probe and the tissue when data are collected, thereby introducing artifacts in the collected signal.…”

Empirical method for rapid quantification of intrinsic fluorescence signals of key metabolic probes from optical spectra measured on tissue-mimicking turbid medium

“…The optical properties of the phantoms were designed based on the formerly reported optical properties of mice skin. 20 Specifically, the tissue-mimicking phantoms had the following average absorption coefficients and reduced scattering coefficients (400 to 600 nm): and . For every single set of absorption and scattering phantom, either 2-NBDG or TMRE was added at biologically relevant concentrations to mimic the fluorophore uptake in tissues.…”