Figure 6: Transmural SM profiling of Crohn’s disease human intestinal specimens.

(a) Transmural surgically resected formalin-fixed (TS-SRFF) sample from colon area deemed uninvolved. SM revealed mesenteric fat/fibrosis, submucosal SM-liquefaction lesion (SmLL), ‘hairline’ penetrating fistulous tracts (PFT) underneath unaffected mucosa, and complex intramural cavernous fistulous tract (IM-CavFT) in deep muscle layers (Supplementary Table 1 and Supplementary Figs 16–19). Histology confirmed inflammation and microscopic purulent material (inset); notice histological tissue distortion. Photo assembled from SM-images. (b) TS-SRFF colon sample from CD-involved area, same patient. SM revealed inter-cobblestone fissures (lines). Manual SM-micro-dissection (SMmD) of submucosa fissure lesion (dashed line). (c) DNA qPCR quantification of 16S bacterial families in SMmD tissues from colon of a. Two sample sets from the same patient (#6–7; 2 cm apart). (d) Cluster analysis (unsupervised, Euclidean) of 16S qPCR-CT values after subtraction (ΔCT) from β-actin for five bacterial families and two universal primers indicated IM-CavFT has distinct flora. Data from a and c and three SAMP tissues (ileum normal, cobblestone; distal colon) indicate SMmD enables SM-lesion-associated flora differentiation. (e) Cluster analysis of 16S qPCR-CT data of colon in a expanded to 13 bacterial families, with duplicated β-actin, and host foxN1 confirms unique flora in IM-CavFT (increased segmented filamentous bacteria Sfibct.1008/clostridia). β-Actin indicates host-cell density. Serosa has distinct flora serving as an internal control to validate enriched/suppressed microbial species and determine contamination occurring during surgical–pathological handling (in vivo healthy serosa should be free of microbial contaminants). (f) TS-SRFF ileum sample from another patient. Notice transmural SmLL glossy lesions originating in inter-cobblestone (dashed line) regions. Scale bar, 1 mm.