Mesoscopic 3D imaging of pancreatic cancer and Langerhans islets based on tissue autofluorescence

Abstract

The possibility to assess pancreatic anatomy with microscopic resolution in three dimensions (3D) would significantly add to pathological analyses of disease processes. Pancreatic ductal adenocarcinoma (PDAC) has a bleak prognosis with over 90% of the patients dying within 5 years after diagnosis. Cure can be achieved by surgical resection, but the efficiency remains drearily low. Here we demonstrate a method that without prior immunohistochemical labelling provides insight into the 3D microenvironment and spread of PDAC and premalignant cysts in intact surgical biopsies. The method is based solely on the autofluorescent properties of the investigated tissues using optical projection tomography and/or light-sheet fluorescence microscopy. It does not interfere with subsequent histopathological analysis and may facilitate identification of tumor-free resection margins within hours. We further demonstrate how the developed approach can be used to assess individual volumes and numbers of the islets of Langerhans in unprecedently large biopsies of human pancreatic tissue, thus providing a new means by which remaining islet mass may be assessed in settings of diabetes. Generally, the method may provide a fast approach to provide new anatomical insight into pancreatic pathophysiology.

Figure 1

Protocols for label free, AF based imaging of pancreatic tissue biopsies. Schematic outlining two alternative protocols for AF based imaging of pancreatic tissue biopsies (see methods for details). The fast track protocol (a) enables 3D analyses of AF features, including generation of full 3D and tomographic data, within in less than 7 h from that the sample is received. In the optimal quality track (b) we attempted to optimize every parameter of the protocol for imaging scenarios for which time is not a critical factor.

Figure 2

AF-based 3D analyses of PDAC biopsies facilitates assessments of tumor delineation and vascularisation. (a–j) Biopsy collected from a patient with PDAC verified by histopathology. (a) Bright field image of uncleared biopsy after fixation. (b) Segmented anatomy (OPT-generated iso-surface reconstruction) of the biopsy based on its endogenous fluorescence. (c) Maximum intensity projection (MIP) view of the tomographic data set in a part of the spectrum enabling detection of vascular and ductal structures. Note the pronounced vascularisation into the PDAC tissue. (d) “Transparent anatomy” segmentation based on tissue AF. The delineation of the tumor tissue is based on low AF intensity regions and appears like a grey mesh that corresponds to histopathological analyses of the same tissue (h–j). (e) Schematic outline of the visualized tumor volume. (f) Tomographic OPT section through the PDAC biopsy seen in (a). (g) LSFM section corresponding to the section seen in (f) of the same specimen. (h) HTEX stained paraffin section of the sample seen in (a) approximately corresponding to the tomographic sections in (f,g). Note, the delineation of the tumor tissue corresponds, section by section, with the outline of the tumor as visualized in (d). (i) Tissue section consecutive to (h), stained for Ck18 as marker for epithelial tissue. (j) Section consecutive to (h,i), stained for aSMA as a marker for major blood vessels. Abbreviations; OPT, optical projection tomography: LSFM, light sheet fluorescence microscopy: HTEX, hematoxylin/eosin; Ck18, cytokeratin 18: aSMA, smooth muscle alpha-actin. Scale bar in (e) is 1500 μm in (a–e), scale bar in (j) is 1000 μm in (f–j) and scale bar in (j′″) is 50 μm in (f′–j′″).

Figure 3

3D imaging of IPMN cyst with associated vessels and islets based on its AF properties. (a–h) A pancreatic cyst from a patient with intraductal papillary mucinous neoplasia. (a) Bright field image of the cyst after fixation. (b) Maximum intensity projection (MIP) view of the tomographic data set in a part of the spectrum enabling detection of vascular and structures. (c) MIP view of the cyst (in a) in the Near infrared (NIR) spectrum illustrating the possibility to visualize the islets of Langerhans based on their AF properties (see also Fig. S1). (d) Tomographic section through the cyst approximately corresponding to the section in (e) showing the cyst wall and associated vessels. (e) HTEX staining through a cross section of the cyst post OPT-imaging. (f) Iso-surface reconstruction of the cyst showing vasculature (red) and islets of Langerhans (green), see also Video S6. (g) Tomographic section through the cyst corresponding to (d) showing the islets of Langerhans. (i) Overlay of the tomographic sections in (d, red) and (g, green). (h) Tissue section consecutive to (e), stained for Ck18 as marker for epithelial tissue. (j) Section consecutive to (e), stained for aSMA as a marker for major blood vessels. (d′,e′,g′–i′), blow up views corresponding to the insets in (d,e,g–j). Abbreviations; Ex, Excitation: Em, Emission: HTEX, hematoxylin/eosin: Ck18, Cytokeratin 18: aSMA, smooth muscle alpha-actin. Scale bar in (c) is 1000 μm in (a–c), scale bar in (i) is 500 μm in (d,g,i), scale bar in (i′) is 100 μm in (d′,g′,i′), scale bar in (j) is 200 μm in (e,h,j), scale bar in (j′) is 100 μm in (e′,h′,j′). Scale bar in (f) is 1000 μm.

Figure 4

Histopathological assessments of PanIN tissue regions can be coupled to mesoscopic 3D assessments based on AF. (a) HTEX staining of a low grade PanIN region from the specimen displayed in Fig. 1. (b) LSFM Sect. (3.8 μm) of the same uncut specimen corresponding to the HTEX section in (a). (c) Larger area encompassing the structure seen in (b, open arrow). (d,e) Shadow projections based on the AF signal seen in (b,c), showing the 3D structure of the PanIN tissue seen in (a). The section plane in (d) corresponds to (c) and the open and closed arrow in (e) corresponds to those in (c). Abbreviations; Ex, Excitation: Em, Emission: HTEX, hematoxylin/eosin. Scale bar in (e) is 50 μm in (a,c–e), scale bar in (b) is 20 μm.

Figure 5

Imaging of islet mass distribution based on endogenous islet AF. (a–f) Images displaying a tissue biopsy from a non-diabetic donor based on their AF properties using indicated filter sets. (a–c) Raw OPT projection view (a), post-processed maximum projection intensity (MIP) image (b) and segmentation of the AF signal delineating tubular structures (c, red). (d–e) Raw OPT projection view (d), post-processed maximum projection intensity (MIP) image (e) and segmentation of the AF signal delineating the Islets of Langerhans (f, green). (g–i) Epifluorescence images of cryosections (20 μm) showing the islet specificity of the AF signal using the indicated filter sets. (g) Unstained control section showing absence of AF in the indicated spectrum. (h) Consecutive section stained for insulin. (i) Unstained consecutive section to (g,h) visualized in the indicated spectrum showing AF specific to the islet, compare i and h. Scale bar in (f) is 500 μm in (a–f), scale bar in (i) is 200 μm in (g–i) and scale bar in (k) is 1000 μm in (j,k).

Figure 6

OPT data of islet volume and number distributions correlate with stereological assessments. (a) Graphs displaying islet mass distribution from OPT measurements (displayed as islet diameters calculated from average length of the individual islets x, y and z axis, green) into size categories of a total of 7034 islets (n = 4 biopsies), compared to stereological assessments as described by Hellman et al., (red). (b) The same stereological data as in (a) but from individual pancreata. (c) The same OPT-data as in (a) but from individual biopsies. (d) The same data as in (a) but displayed as the number of islets falling within each size category. (e) The same stereological data as in (d) but from individual pancreata. (f) The same OPT-data as in (a) but from individual biopsies. Notwithstanding that the OPT material was collected from different regions of the pancreata (see methods), it is well in line with previous stereological assessments. The data in (a,d) are presented as mean ± SEM.