Experimental design
Figure 1 shows the imaging workflow. The full analysis is done on a sample of tumor and adjacent non-tumorous tissue obtained from the fresh resection specimen, ex vivo, shortly after surgery. The key steps are rapid fixation/dehydration in acetone, fast high-resolution phase-contrast microtomography, and image processing and visualization. Finally, we compare the x-ray histology with classical histology on the same samples. Below we outline the steps. More details can be found in “Materials and methods”.
Fig. 1
Workflow for x-ray and classical histology of solid tumor. (a) The tumor, including a margin of healthy tissue, is surgically removed from the patient and sliced in ~ 5 mm slabs. (b) Fixation in 100% acetone. (c) 3D x-ray histology by phase-contrast microtomography. (d) Standard histological sample preparation, including formalin fixation, dehydration, paraffin embedding, sectioning and staining. (e) Light microscopy of H&E-stained slice.
Rapid acetone fixation/dehydration
A sample of the fresh tumor from the surgery is placed in 100% acetone. A typical sample size is 5 × 25 × 25 mm (liver, pancreas) as well as smaller diagnostic biopsies (sarcomas). Acetone is known for its faster penetration of tissue compared to ethanol and methanol. By exchanging the classical formaldehyde fixation step followed by a series of dehydration steps in increasing concentration of alcohol for a single-step acetone fixation/dehydration step we can reduce the preparation time from a few days to a few hours before the sample is ready for imaging. Depending on tissue type, the morphology may be slightly more affected compared to when using the gentler method of gradually increasing alcohol concentrations. However, for the purpose here (i.e., to determine if the resection margin is free of tumor cells) this limitation is not a major concern: the morphological changes are relatively small, allowing reliable assessment of the tumor border. Tissue shrinkage is discussed in more detail in the Supplementary Material.
Phase-contrast x-ray microtomography
The fixed and dehydrated sample is placed in the laboratory propagation-based phase-contrast imaging arrangement (cf. Fig. S1)15,16,19. It consists of an x-ray source (125W, 70 kV microfocus liquid–metal-jet source), a precision rotation stage and a 4096 × 4096 pixel CMOS detector with a 10 µm Gadox scintillator. Imaging is performed with 2 × geometric magnification, resulting in a pixel resolution of 4.5 µm and a FWHM blur of 11 µm. The effective distance zeff was adjusted to obtain maximum contrast for cellular-sized structures15, while still keeping the exposure times reasonable (2–3 h) with the present low-power source.
Image processing and visualization
This includes initial image corrections, phase-retrieval, tomographic reconstruction, different artifacts and noise reduction routines, and finally displaying the full 3D volume. From this 3D volume virtual histology slices are extracted. Typical processing time is 1–2 h.
Classical histology
After the x-ray imaging, the acetone-fixed samples were fixed in formaldehyde 4% buffered solution, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E) according to standard procedure, after which a classical histopathology assessment was performed by a specialized pathologist.
Comparison
Finally, the x-ray histology and the classical histology of the same sample were compared. Due to the different states of the sample in the two imaging modalities (fixed tissue slab versus micrometer-thin slice), the classical histology slice does not exactly overlap with the virtual x-ray tomography slice, but it is close enough for a relevant comparison.
The method has been investigated for several types of tumors. Below we present results for increasingly difficult tumor morphologies, from liver over pancreas to sarcomas.
Case series
A total of 12 tumors from 12 individual patients were analyzed, comprising surgical resection specimens of colorectal liver metastases (n = 3), hepatocellular carcinoma (n = 3), intrahepatic cholangiocarcinoma (n = 1), pancreatic neuroendocrine tumor (n = 1), pancreatic ductal adenocarcinoma (n = 1), and diagnostic biopsy probes of chondrosarcoma (n = 1), soft-tissue sarcoma (n = 1), and osteosarcoma (n = 1). Below we present results from 2 types of liver cancer, 2 types of pancreatic cancer and 2 types of sarcomas. Data from all remaining samples is available in Supplementary Material (Figs. S2–S7). Full 3D stacks are available on request.
Liver tumors
Here we show the results for two common types of liver tumors: Colorectal liver metastasis (n = 3) and hepatocellular carcinoma (n = 3). Intrahepatic cholangiocarcinoma (n = 1) is presented in the Supplementary Material (Fig. S4).
Figure 2 compares the x-ray histology and the classical histology for the colorectal liver metastasis. Colorectal cancers are the second most deadly cancer type24. Approximately 30% of the patients affected develop the liver metastasis investigated here.
Fig. 2
Colorectal liver metastasis. (a) Maximum-intensity projection (MIP) virtual slice through the microtomography volume, matching (b) the classical histology slice (H&E staining). The sample contains normal liver (L) and tumor (T). (c) Liver tissue, as seen with x-ray histology (left) and classical histology (right). (d) Tumor periphery, with fibrous septa (Fi) and necrotic foci (N). (e) Portal zone, with vein (V), artery (Ar) and bile duct (BD). (f) Tumor (T), necrosis (N) and fibrosis (Fi) in the central region of the metastasis. Scale bars: 1 mm.
Figure 2a,b show a virtual slice of the phase-contrast x-ray microtomography (a) that matches the classical histology slice (b). The tumor tissue (right) is easily distinguishable from the normal liver tissue (left). In Fig. 2c the normal liver tissue is shown for the two methods. The appearance is similar but with somewhat larger graininess in the x-ray histology. Figure 2d shows the tumor periphery for the two methods. In addition to its characteristic large glandular pattern, fibrotic septa and foci of necrosis are observable with both methods. Figure 2e shows a medium-sized portal zone with vein, artery and the bile duct branches (portal triad). Finally, Fig. 2f shows a region towards the metastasis center where scarce tumor, extensive necrosis, and fibrosis all appear within a 3 × 3 mm area. All main tissue components of the colorectal liver metastasis are observable in the x-ray as well as the classical histology. We tested 3 samples from three different patients, all showing a good visual agreement between x-ray and classical histology.
Figure 3 compares the x-ray histology and the classical histology for hepatocellular carcinoma. Primary liver and bile duct cancers are the 3rd most deadly cancer type24. Hepatocellular carcinoma, here investigated, accounts for 75% of the primary liver tumors.
Fig. 3
Hepatocellular carcinoma. (a) Virtual slice (MIP) through the microtomography volume, matching (b) the classical histology slice (H&E staining). The sample contains normal liver (L) and tumor (T). (c) Virtual slice through the microtomography volume, corresponding to the green dashed line in (a). The blue dashed line marks the position of the slice in (a). Fibrous septa (Fi) within and encapsulating the tumor and the hepatic vein (HV) can be seen. (d) Close-up of tumor (T) with intervening fibrotic septa (Fi) in x-ray (left) and classical histology (right). Scale bars: 1 mm.
Figure 3a,b shows a virtual slice of the phase-contrast x-ray microtomography (a) that matches the classical histology slice (b). Both normal liver as well as tumor tissue are easily distinguishable in both images. In addition, we observe intratumoral fibrotic septa, encapsulating fibrosis and a medium sized hepatic vein. Figure 3c shows a section perpendicular to (a), to illustrate the 3D capacity of the x-ray tomography. Here we also observe tumor tissue, normal liver tissue, fibrous septa and encapsulations, and even a continuous longitudinal section along a hepatic vein. Finally, Fig. 3d shows a 4 × 4 mm close-up region of the tumor center with the septal fibrous pattern clearly observable in both modalities. Again, we tested 3 samples of hepatocellular carcinoma from three different patients with comparable results.
Pancreas tumors
Here we show the results for the most common types of pancreatic cancer: Pancreatic ductal adenocarcinoma (n = 1) and the rare pancreatic neuroendocrine tumor (n = 1).
Figure 4 compares the x-ray histology and the classical histology for the pancreatic ductal adenocarcinoma. This cancer type accounts for 90% of the pancreatic cancers. It has a poor prognosis, with a five-year survival rate of 9% worldwide25.
Fig. 4
Pancreatic ductal adenocarcinoma. (a) Virtual slice through the microtomography volume, matching (b) the classical histology slice (H&E staining). The sample contains normal pancreatic tissue (P) and a region of intermixed fibrosis (Fi) and tumor. Pancreatic ducts (PD) and interlobular fat (F) can also be seen. (c) Desmoplastic fibrosis in micro-CT (left) and classical histology (right). (d) Large, irregular tumor glands (T) in x-ray (left) and classical histology (right). Scale bars 1 mm.
Figure 4a,b shows a virtual slice of the phase-contrast x-ray microtomography (a) that matches the classical histology slice (b). We observe normal pancreatic as well as tumor tissue. In addition, fibrotic areas and ensembles of individual fat cells are visible, as well as the pancreatic ducts. Figure 4c shows that the desmoplastic fibrosis in which the tumor tissue is embedded is clearly visible also in the x-ray tomography. Finally, in Fig. 4d we observe infiltrating, large, irregular tumor glands near the edge of the tissue sample.
Figure 5 compares the x-ray histology and the classical histology for the pancreatic neuroendocrine tumor. This tumor type is less common and accounts for 1–2% of the pancreatic cancers26.
Fig. 5
Pancreatic neuroendocrine tumor. (a) Virtual slice through the microtomography volume, matching (b) the classical histology slice (H&E staining). The sample contains peripancreatic fat (F), neuroendocrine tumor (T) and a lymph node metastasis (LNM). (c) Close-up of the tumor (T), in ring-like configuration, with blood (Bl). (d) Lymph node metastasis (LNM), fat (F) and vein (V) in the peripancreatic soft tissue. Scale bars: 1 mm.
Figure 5a,b shows a virtual slice of the phase-contrast x-ray microtomography (a) that matches the classical histology slice (b). We observe fat tissue, tumor tissue, and a lymph node metastasis, all clearly delineated. Figure 5c shows at high magnification a microscopic area where the tumor cells are configurated in a ring-like structure filled with blood. Finally, Fig. 5d shows a close-up of the lymph node metastasis in the peripancreatic soft tissue.
Sarcomas
Here we show the results from two different sarcomas. Sarcoma is a rare, aggressive and heterogeneous group of tumors that arise throughout the body. The incidence is approximately 10% of pediatric cancers and 1% of all adult malignancies27. The value of resection margin for local control of the disease is well established for this group of cancers28,29. However, their challenging growth pattern makes resection margin assessment difficult, pointing at the present limitations of our technique. A chondrosarcoma is presented in Supplementary Material (Fig. S7).
Figure 6 compares the x-ray histology (a) and the classical histology (b) for an osteosarcoma. The phase-contrast image differentiates viable tumor tissue adjacent to an area of necrosis and healthy fat tissue. Collagen-rich fibrous tissue, blood, and a blood vessel can also be distinguished. Closer histopathological examination of the H&E section at a higher magnification confirmed the boundaries between tumor and necrotic tissue (c) and the presence of collagen fibers between the tumor and adjacent fat tissue (d).
Fig. 6
Osteosarcoma. (a) Virtual slice from the microtomography of a tumor biopsy matching (b) the H&E-stained section. The images show tumor tissue (T), a region of necrosis (N) delineated in yellow , blood (Bl) a blood vessel (V) and fat (F) with hemorrhagic fibrous septa (Fi). The lower panel shows magnified images in x-ray histology (left) and classical histology (right) of (c) the tumor-necrosis boundary. (d) Hemorrhagic connective fibrous tissue (Fi) between the tumor and the adjacent fat tissue. Scale bars: 1 mm.
Figure 7 compares the x-ray histology (a) and the classical histology (b) for a low-grade fibromyxoid sarcoma, a type of soft-tissue malignancy. The tumor is characterized by a bland spindle cell population within an alternating fibrotic and myxoid stroma. In Fig. 7c, a close-up of the tumor border is shown, visible in both the x-ray and the classical histology. Figure 7d shows a non-tumorous, overlying region of fibroadipose tissue and skin.
Fig. 7
Low-grade fibromyxoid sarcoma. (a) Virtual slice from the x-ray microtomography of a tumor biopsy matching (b) the H&E-stained histology section. The tumor is characterized by a bland spindle cell population within an alternating fibrotic and myxoid stroma, with poorly circumscribed borders. The lower panels show the x-ray histology and the classical histology of (c) a closeup of the tumor border (yellow dashed line) with collagen-rich fibrosis (Fi) visible in the x-ray histology image and (d) overlying skin (S) and fat lobules (F). Scale bars: 1 mm.
