Early tumor development captured through nondestructive, high resolution differential phase contrast X-ray imaging

Abstract

Although a considerable amount is known about molecular dysregulations in later stages of tumor progression, much less is known about the regulated processes supporting initial tumor growth. Insight into such processes can provide a fuller understanding of carcinogenesis, with implications for cancer treatment and risk assessment. Work from our laboratory suggests that organized substructure emerges during tumor formation. The goal here was to examine the feasibility of using state-of-the-art differential phase contrast X-ray imaging to investigate density differentials that evolve during early tumor development. To this end the beamline for TOmographic Microscopy and Coherent rAdiology experimenTs (TOMCAT) at the Swiss Light Source was used to examine the time-dependent assembly of substructure in developing tumors. Differential phase contrast (DPC) imaging based on grating interferometry as implemented with TOMCAT, offers sensitivity to density differentials within soft tissues and a unique combination of high resolution coupled with a large field of view that permits the accommodation of larger tissue sizes (1 cm in diameter), difficult with other imaging modalities.

FIG. 1

TOMCAT end-station with the grating interferometer setup for differential phase contrast imaging, as described by McDonald et al. (11). A container holding the samples is placed in phase-matching milliQ-water in the path of the X-ray beam (indicated by the redline arrow). The phase shift is measured with the grating interferometer together with a grating-stepping protocol (22). The piezo stage controls the grid translation for phase stepping, measuring the distribution of refraction angles imposed by the object being scanned.

FIG. 2

Representative H&E stained tumor sections and corresponding raw and rendered differential phase contrast X-ray images of the Lewis lung carcinoma (LLC) tumors sacrificed at different time points. All H&E images are a series of stitched images obtained with a 10× objective. Panel a: H&E of section of LLC tumor growing in C57BL/6 mouse sacrificed at the earliest time point, T = 0 days, when the tumor was first apparent. Panel b: The average tumor volumes for the tumors as a function of time point of sacrifice. Panel c: H&E of LLC tumor sacrificed at 3 days, T = 3 days after first measurable tumor, middle time point. The tumor area within the tissue is outlined in red. Considerable intra-tumor substructure is detected. Note, substantial regional differentials in H&E staining, with white, pink and purple banding, indicative of organization of cellular and nuclear material within the tumor boundary. Panel d: An image slice from the differential phase contrast (DPC) X-ray image obtained by TOMCAT for the same T = 3 day tumor as the H&E section of panel c. Panel e: Processed image providing a pseudo-color gradient based on density of tissue from the phase contrast images. Panel f: H&E of LLC tumor sacrificed at 6 days, T = 6, after first measurable tumor, a late time point. As in panel c, the tumor area is outlined in red. The intra-tumor compartmentalization and substructure seen at T = 3 has now evolved and is resolving despite the fact the tumor has grown in size and infiltrated fat and muscle tissue. Panel g: A series of DPC images representing every 20 slices for the LLC tumor shown in panels f and h. Panel h: An image slice from the differential phase contrast (DPC) X-ray image obtained by TOMCAT for the same T = 6 day tumor as the H&E section of panel f. Yellow outlines enclose examples of substructure regions determined by density changes in the tissue. The black arrows point to the skin and blue arrows to the fat tissue. The gray scale bar depicts the range of gray values in the DPC images. Select tissue regions (fat, muscle and skin) have been associated with specific gray values in DPC images. Panel i: Processed image providing a pseudo-color gradient based on density of tissue from the phase contrast images. Scale bars are 500 µm.

FIG. 3

Representative differential phase contrast X-ray image and 3D processed image for LLC tumors. Panels a and b: Orthogonal views from an image slice of the DPC image. Red outlines enclose substructures determined by density changes in the tissues, with inserts providing enlarged views of these regions. Panel c: One method of 3D rendering with pseudo-coloring from the phase contrast images with slices from panels b and c within the tissue to indicate orientation of the slice for T = 3 days. Panel d: Close-up of the tumor as rendered from phase contrast images for T = 3 days, separating the tumor tissue from the attached adjacent microenvironment to highlight differential density structures within each tissue. Scale indicates the tumor diameter to be 3.9 mm.