We propose here a revision of the anatomical concepts of the submucosa, dermis, fascia, and vascular adventitia, suggesting that, rather than being densely-packed barrier-like walls of collagen, they are fluid-filled interstitial spaces. The presence of fluid has important implications for tissue function and pathology. Our data comparing rapidly-biopsied and frozen tissue with tissue fixed in a standard fashion suggest that the spaces we describe, supported and organized by a collagen lattice, are compressible and distensible and may thus serve as shock absorbers. All of the organs in which we have detected this structure are subject to cycles of compression and distension, whether relatively constant (lungs, aorta) or intermittent (digestive tract after a meal, urinary bladder during micturition, skin under mechanical compression, fascial planes during action of the musculoskeletal system). The dermal interstitium and the fascial interstitium may be mechanistically important in explaining edema (as with the pre-obstructed bowel in Supplemental Fig. 2). “Third spacing” in post-operative lymphedema (as when draining nodes are excised) and anasarca due to liver, renal, or cardiac failure may reflect fluid distention and stasis in this interstitial space. The submucosal interstitial space of the biliary tree – extending through the full extent of the extra- and intra-hepatic portal stroma – may explain the characteristic duct edema that rapidly develops in acute large bile duct obstruction.

Further support for the relationship between the histology we observe and in vivo structure comes from ultrasonography of tissues. Endoscopic ultrasound of the bile duct shows that it consists of three layers, the middle one of which comprises 90% of the wall thickness and is fluid filled7; it corresponds to the submucosal interstitium. Submucosal spaces of other viscera, the dermis, and fascia appear heterogeneous by ultrasound, typically indicative of fluid or adipose tissue, while truly dense collagenized stroma, such as in tendons and ligaments, appears dark by ultrasound8,9,10,11. Additional support for our observations is found in published ultrastructural studies of skin, vermiform appendix, and peri-aortic adventitia, which also appear to have included these structures, although they were not well characterized12,13,14. In the liver, the previously identified “space of Mall” in the portal region may represent this interstitium15. Indeed, Mall’s original drawings, derived from injection studies, appear to represent the same structures we identify here16.

The nature of the lining cells is unclear. While cells of the extrahepatic bile duct stained for both CD34 and D2-40, D2-40 staining was absent in all other tissues examined. Vascular endothelial cells also co-express CD34 and vimentin, but the lack of endothelial features on electron microscopy excludes this classification for the interstitial lining cells we observe, suggesting instead that they are a novel, CD34-positive form of fibroblast or even mesenchymal stem cell17. Whether they are the cells that deposit the collagen bundles is unknown; if so, they would be important in scar formation in wound healing. Notably, keloid scars show collagen bundles and large spaces that appear to be an exaggeration of these structures in the underlying dermis18. Recent data showing that keloid scars appear in regions of skin under high tension raise questions about the impact of mechanical forces and fluid flow on the structures and cells of this space19.

It is likely that the submucosal interstitium we describe corresponds to the interstitial spaces described in studies of metastasizing cell clusters20. The presence of a network of submucosal channels in the digestive and urinary tracts could explain the greatly increased likelihood of metastasis by luminal invasive tumors once they reach the submucosa. As illustrated by the cases we show of invasive melanoma and gastric cancer (Fig. 4D–I), the presence of submucosal/dermal fluid-filled channels also suggests the reason for T2 lesions being at such significantly increased risk for metastasis over stage T1 lesions – because visceral submucosae and the dermis are open, fluid filled spaces, rather than a wall of dense of connective tissue, they may be easily traveled by invasive tumor cells. Moreover, the mechanical pressure on such spaces (peristalsis in the digestive tract, compression and/or movement-associated pressure on skin) could further promote spread through these spaces. If the interstitial lining cells are the precursors of fibrogenic myofibroblasts, they might also function as first responders in peri-tumoral sclerosis of the pancreatico-biliary tree, tubular digestive tract, bronchial tree, urinary bladder and skin. Indeed, a unique population of CD34/vimentin co-expressing peritumoral fibroblasts has been reported21. These cells could also serve important roles in non-malignant sclerotic conditions including biliary atresia and primary sclerosing cholangitis in the biliary tree, scleroderma in the dermis and esophagus, and inflammatory bowel disease in the digestive tract. Ongoing studies are focused on characterizing these cells and their functions.

The flow of interstitial fluid through the submucosal space of the luminal GI tract is likely guided by peristalsis, in parallel with luminal contents. If there is communication between the gut lumen and the submucosal space, this raises the possibility that cell signaling (including hormonal or immunologic signals) could be regulated in a proximal-to-distal manner determined by the speed of peristalsis. Immunologic interactions in this interstitial space could also be important in inflammatory conditions such as primary sclerosing cholangitis, chronic pancreatitis, inflammatory bowel disease and scleroderma. Interestingly, while macrophages were not seen in these spaces in the normal tissues examined, they clearly traffic into the space to take up the tattoo pigment after submucosal injection (Fig. 4A–C).

The collagen bundles in the interstitial space are lined on only one side by cells, implying that the collagen matrix on the opposite side is in direct contact with interstitial fluid. There are few examples known in the human body other than the interstitial space between cells, the renal glomerulus and the space of Disse, where fluid is in direct contact with matrix proteins without an intervening cell barrier. Collagen fibers, which are charged molecules, may form an important physiologically active surface. Whether cells of the immune system or other cells passing through the space interact with the collagen bundles is a highly physiologically relevant question that requires further investigation.

In sum, while typical descriptions of the interstitium suggest spaces between cells, we describe macroscopically visible spaces within tissues – dynamically compressible and distensible sinuses through which interstitial fluid flows around the body. Our findings necessitate reconsideration of many of the normal functional activities of different organs and of disordered fluid dynamics in the setting of disease, including fibrosis and metastasis. A submucosa subjected to directional, peristaltic flow is not the previously envisaged wall of dense connective tissue, but a potential conduit for movement of injurious agents, pro-fibrogenic signaling molecules, and tumor cells. This raises the possibility that direct sampling of the interstitial fluid could be a diagnostic tool. Finally, our study demonstrates the power of in vivo microscopy to generate fresh insights into the anatomy and physiology of normal and diseased tissues.