This document discusses the discovery of a "new organ" called the interstitium. Through the use of probe-based confocal laser endomicroscopy, a reticular pattern was observed in the submucosa of the bile duct and pancreatic duct. Further investigation revealed that this space is a cavernous interstitial sinus, lined by a novel fibroblast/endothelial hybrid cell and structured by a collagen bundle lattice. Similar structures were also found in the dermis, digestive tract submucosa, urinary bladder submucosa, and other tissues. This interstitial space may have implications for metastasis, sclerosis, and other processes. The discovery challenges traditional definitions of tissues and suggests the inter

1. Neil Theise, MD
Department of Pathology
New York University School of Medicine
New York City
Discovering a “New Organ”
Tales of the Interstitium

2. PROLOGUE

3. “Thinking is better than knowing,
but looking is even better.”
Goethe, “Maxims”

4. Goethe, “Maxims”
“Thinking is better than knowing,
but looking is even better.”

5. A fundamental question:

6. What is the body made of?
Before microscopy there was philosophy
Two models from Ancient Greece:
Is the body made of indivisible subunits?
or
Is the body an endlessly divisible fluid continuum?
A fundamental question:A fundamental question:

7. What is the body made of?
Before microscopy there was philosophy
Two models from Ancient Greece:
Is the body made of indivisible subunits?
or
Is the body an endlessly divisible fluid continuum?
A fundamental question:

8. What is the body made of?
Before microscopy there was philosophy
Two models from Ancient Greece:
Is the body made of indivisible subunits?
or
Is the body an endlessly divisible fluid continuum?
A fundamental question:
Thinking/Knowing Looking

9. What is the body made of?
Before microscopy there was philosophy,
Two models from Ancient Greece:
Is the body made of indivisible subunits?
or
Is the body an endlessly divisible fluid continuum?

10. What is the body made of?
Before microscopy there was philosophy,
Two models from Ancient Greece:
Is the body made of indivisible subunits?
or
Is the body an endlessly divisible fluid continuum?

11. R. Hooke (1665)
:
Leeuwenhoek's microscopes
Henry Baker (1739)

14. Microscopy through history:
manipulating and understanding
ARTIFACTS

15.  Leeuwenhoek in 1719 – saffron for muscle fiber
 Goppert and Cohn in 1849 – carmine
 Gerlach in 1858 – selective nuclear staining
 Waldeyer in 1863 - hematoxylin
Artifacts from staining…
Heating
Chemical fixation
Freezing
Dehydration
Oxidation
Artifacts from fixing…

20.  Leeuwenhoek in 1719 – saffron for muscle fiber
 Goppert and Cohn in 1849 – carmine
 Gerlach in 1858 – selective nuclear staining
 Waldeyer in 1863 - hematoxylin
Artifacts from staining…
 Heating
 Chemical fixation
 Freezing
 Dehydration
 Oxidation
Artifacts from fixing…

21.  Leeuwenhoek in 1719 – saffron for muscle fiber
 Goppert and Cohn in 1849 – carmine
 Gerlach in 1858 – selective nuclear staining
 Waldeyer in 1863 - hematoxylin
Artifacts from staining…
 Heating
 Chemical fixation
 Freezing
 Dehydration
 Oxidation
Artifacts from fixing…

23. The story, Chapter 1
A pathologist and
an endoscopist
walk into a bar…?

24. What is Optical Biopsy ? 24
Physical Biopsy
Optical Biopsy
En-face view
In-vivo
Microscopic
Minimally invasive
Instantaneous imaging
24
Transverse view
Ex-vivo
Microscopic
Invasive
Delayed imaging

25. Probe based Confocal Laser Endomicroscopy
How it works:
 Fluorescein: extracellular contrast
 492 nm absorption spectrum
 Depth of focus; ~70 mM

26. pCLE fixed focal depth
Esophagus

27. pCLE fixed focal depth
Stomach

28. pCLE fixed focal depth
Small Intestine

29. pCLE fixed focal depth
Large Intestine

30. But in the biliary and pancreatic ducts,
something weird appears by pCLE…
The “reticular pattern.”

31. Normal Common Bile Duct

32. Normal Common Bile Duct

33. Normal Common Bile Duct

34. Normal Common Bile Duct

35. Normal Common Bile Duct
 Submucosal capillaries?
 Arteries? Veins?
 Muscularis mucosae?
 Mucosal crypts?
 Peribiliary glands?

46. Real spaces
NOT artifactual spaces!

47. The submucosa of the
bile duct is a:
 Cavernous lymphatic?
space;
 Structured by a collage
bundle lattice.

48. The submucosa of the
bile duct is a:
 Cavernous lymphatic?
space;
 Structured by a collage
bundle lattice.

49. The submucosa of the
bile duct is a:
 Cavernous lymphatic?
space;
 Structured by a collage
bundle lattice.
FITC appears simultaneously
in lymph nodes & submucosa,
20-30 sec
after vascular filling.

50. CD34 Vimentin
frozen frozen
fixed fixed

52. The submucosa of the common bile duct (and pancreatic duct) is a:
 Cavernous lymphatic? sinus;
BUT: the CD34 makes this difficult,
AND: its not a vessel, but a sinus…
But the fluid seems to be LYMPH!
Structured by a collage bundle lattice;
 Collagen bundles are made of types 1/3 collagen (tbc);
 Lined on one side by a novel fibroblast/endothelial hybrid cell;
 The opposite side from which is a direct matrix:fluid interface.

53. The submucosa of the common bile duct (and pancreatic duct) is a:
 Cavernous lymphatic? sinus;
BUT: the CD34 makes this difficult,
AND: its not a vessel, but a sinus…
But the fluid seems to be LYMPH!
Structured by a collage bundle lattice;
 Collagen bundles are made of types 1/3 collagen (tbc);
 Lined on one side by a novel fibroblast/endothelial hybrid cell;
 The opposite side from which is a direct matrix:fluid interface.
Wiki:
Lymph is the fluid that circulates throughout the lymphatic system. The
lymph is formed when the interstitial fluid (the fluid which lies in the interstices of all
body tissues) is collected through lymph capillaries.
It is then transported through lymph vessels to lymph nodes before
emptying ultimately into the right or the left subclavian vein, where it mixes back with
blood.
Since the lymph is derived from the interstitial fluid, its composition
continually changes as the blood and the surrounding cells continually exchange
substances with the interstitial fluid.

54. The submucosa of the common bile duct (and pancreatic duct) is a:
 Cavernous interstitial sinus
Structured by a collage bundle lattice;
 Collagen bundles are made of types 1/3 collagen (tbc);
 Lined on one side by a novel fibroblast/endothelial hybrid cell;
 The opposite side from which is a direct matrix:fluid interface.

55. The submucosa of the common bile duct (and pancreatic duct) is a:
 Cavernous interstitial sinus;
 Structured by a collage bundle lattice;
 Collagen bundles are made of types 1/3 collagen (tbc);
 Lined on one side by a novel fibroblast/endothelial hybrid cell;
 The opposite side from which is a direct matrix:fluid interface.

56. The submucosa of the common bile duct (and pancreatic duct) is a:
 Cavernous interstitial sinus;
 Structured by a collage bundle lattice;
 Lined on one side by a novel fibroblast/endothelial hybrid cell;
 The opposite side from which is a direct matrix:fluid interface.

57. The submucosa of the common bile duct (and pancreatic duct) is a:
 Cavernous interstitial sinus;
 Structured by a collage bundle lattice;
 Lined on one side by a novel fibroblast/endothelial hybrid cell;
 The opposite side of which is a direct matrix:fluid interface.

59. Implications
 Invasion into submucosa potentiates metastasis
 Circumferential compression:
• outward, by impacted stone or tumor
• inward, by tumor or nodes
 Sclerosis (e.g. PSC, biliary atresia) activation of
endothelial/fibroblastic cells?

60. Implications
 Invasion into submucosa potentiates metastasis
 Circumferential compression:
• outward, by luminal tumor or stone
• inward, by extrinsic tumor or nodes
 Sclerosis (e.g. PSC, biliary atresia) activation of
endothelial/fibroblastic cells?

61. Implications
 Invasion into submucosa potentiates metastasis
 Circumferential compression:
• outward, by luminal tumor or stone
• inward, by extrinsic tumor or nodes
 Sclerosis (e.g. PSC, biliary atresia) activation of
endothelial/fibroblastic cells?
Intrahepatic features
of LBDO

62. Implications
 Invasion into submucosa potentiates metastasis
 Circumferential compression:
• outward, by luminal tumor or stone
• inward, by extrinsic tumor or nodes
 Bile duct scarring in inflammatory diseases

63. The story, Chapter 2
Is that all there is…?

65. 10x CD34, 40x

66. Digestive Tract: Implications
 Submucosal compressibility creates “shock absorber” functionality
 Invasion into submucosa potentiates metastasis.
 Lymphatic flow parallels luminal flow: communication? Other
functionalities?
 Tumor invasion into submucosa potentiates metastasis
 Endothelial/fibroblastic cells may mediate peri-tumoral sclerosis.
 Sclerosis (e.g. PSC, biliary atresia) activation of
endothelial/fibroblastic cells?

67. Pre-obstructed bowel (incarcerated hernias)
 Specimens from 6 patients

68. Pre-obstructed bowel (incarcerated hernias)
 Immunostains positive for plasma proteins:
IgG, IgM, IgA, C-reactive protein, albumin, A1AT

69. Pre-obstructed bowel (incarcerated hernias)
 Immunostains positive for plasma proteins:
IgG, IgM, IgA, C-reactive protein, albumin, A1AT
Lymph!

73. Therefore this space links to the lymphatics.

74. Ulcerated surface CA of stomach
Invasive
tumor

75. Ulcerated surface CA of stomach
Invasive
tumor

78. The story, Chapter 3
How thick skinned
is the average pathologist?

81. PREDICTS

82. Skin: Implications
Persistence of tattoos indicates collagen bundles STABLE,
with little to no turn over in normal circumstances…

83. Dermal invasion of melonoma

86. Skin: Implications
 Invasion into dermis potentiates metastasis
 Sclerosis of dermal lymphatic plexus = scleroderma?
 Failure of continual glucose sensors?

87. Skin: Implications
 Invasion into dermis potentiates metastasis
 Sclerosis of dermal interstitial sinus = scleroderma?
 Failure of continual glucose sensors?

88. Skin: Implications
 Invasion into dermis potentiates metastasis
 Sclerosis of dermal interstitial sinus = scleroderma?
 Acupuncture…?

89. The story, Chapter 4
The road goes ever on

90. Seen in:
Perivascular stroma

91. Seen in:
Urinary bladder submucosa

92. Seen in:
Bronchial submucosa and
peri-cartilagenous soft tissue

93. Seen in:
Fascia

94. CNS PNS - Perineurium
Periglandular connective tissue Endocardium

95. Every “densely collagenized” structure of the body…

96. Tendon Ligament

97. Is this interstitium? (what is interstitium?)
Is this fascia? (what is fascia?)
“The fascial system consists of the three-dimensional continuum of soft,
collagen containing, loose and dense fibrous connective tissues that
permeate the body. It incorporates elements such as adipose tissue,
adventitiae and neurovascular sheaths, aponeuroses, deep and
superficial fasciae, epineurium, joint capsules, ligaments,
membranes, meninges, myofascial expansions, periostea,
retinacula, septa, tendons, visceral fasciae, and all the intramuscular and
intermuscular connective tissues including endo-/peri-/epimysium. The
fascial system surrounds, interweaves between, and interpenetrates all
organs, muscles, bones and nerve fibers, endowing the body with a
functional structure, and providing an environment that enables all body
systems to operate in an integrated manner.”
Stecco C, Adstrum S, Hedley G, Schleip R, Yucesoy CA
Update on fascial nomenclature. J Bodyw Mov Ther. 2018; 22: 354.

98. Is this interstitium? (what is interstitium?)
Is this fascia? (what is fascia?)
“The fascial system consists of the three-dimensional continuum of soft,
collagen containing, loose and dense fibrous connective tissues that
permeate the body. It incorporates elements such as adipose tissue,
adventitiae and neurovascular sheaths, aponeuroses, deep and
superficial fasciae, epineurium, joint capsules, ligaments,
membranes, meninges, myofascial expansions, periostea,
retinacula, septa, tendons, visceral fasciae, and all the intramuscular and
intermuscular connective tissues including endo-/peri-/epimysium. The
fascial system surrounds, interweaves between, and interpenetrates all
organs, muscles, bones and nerve fibers, endowing the body with a
functional structure, and providing an environment that enables all body
systems to operate in an integrated manner.”
Stecco C, Adstrum S, Hedley G, Schleip R, Yucesoy CA
Update on fascial nomenclature. J Bodyw Mov Ther. 2018; 22: 354.
But what about dermis and visceral submucosae?

99. Carla Stecco MD, ... Raffaele De Caro MD,
inFunctional Atlas of the Human Fascial
System, 2015

100. Carla Stecco MD, ... Raffaele De Caro MD,
inFunctional Atlas of the Human Fascial
System, 2015

101. Carla Stecco MD, ... Raffaele De Caro MD,
in Functional Atlas of the Human Fascial
System, 2015

102. Prof. Jean Claude Guimberteau

103. Prof. Jean Claude Guimberteau

105. Epilogue

106. Submitted to:
Nature
Nature Medicine
Science
Nature Communications
PLoS 1
PNAS
Journal of Pathology
Rejected without review:
“Not of interest to a general audience.”

107. Submitted to:
Nature
Nature Medicine
Science
Nature Communications
PLoS 1
PNAS
Journal of Pathology
Rejected without review:
“Not of interest to a general audience.”

108. Submitted to:
Nature
Nature Medicine
Science
Nature Communications
PLoS 1
PNAS
Journal of Pathology
Rejected without review:
“Not of interest to a general audience.”

109. Submitted to:
Nature
Nature Medicine
Science
Nature Communications
PLoS 1
PNAS
Journal of Pathology
Rejected without review:
“Not of interest to a general audience.”

110. Submitted to:
Nature
Nature Medicine
Science
Nature Communications
PLoS 1
PNAS
Journal of Pathology
Rejected without review:
“Not of interest to a general audience.”

111. Submitted to:
Nature
Nature Medicine
Science
Nature Communications
PLoS 1
PNAS
Journal of Pathology
Rejected without review:
“Not of interest to a general audience.”

112. Submitted to:
Nature
Nature Medicine
Science
Nature Communications
PLoS 1
PNAS
Journal of Pathology
Rejected without review:
“Not of interest to a general audience.”

113. Submitted to:
Nature
Nature Medicine
Science
Nature Communications
PLoS 1
PNAS
Journal of Pathology
Rejected without review:
“Not of interest to a general audience.”

114. Submitted to:
Nature
Nature Medicine
Science
Nature Communications
PLoS 1
PNAS
Journal of Pathology
Rejected without review:
“Not of interest to a general audience.”

115. Submitted to:
Nature
Nature Medicine
Science
Nature Communications
Public Library of Science 1
Proceedings of the National Academy of Sciences
Journal of Pathology
Rejected without review:
“Not of interest to a general audience.”

116. Submitted to:
Nature
Nature Medicine
Science
Nature Communications
PLoS 1
PNAS
Journal of Pathology
Scientific Reports
Rejected without review:
“Not of interest to a general audience.”

117. Submitted to:
Nature
Nature Medicine
Science
Nature Communications
PLoS 1
PNAS
Journal of Pathology
Scientific Reports
Reviewer 1: “Who would have guessed that there would be new
anatomy to be discovered in 2017? Clear, concise and well written.”

118. Submitted to:
Nature
Nature Medicine
Science
Nature Communications
PLoS 1
PNAS
Journal of Pathology
Scientific Reports
Reviewer 2: “Trivial result. Just an attempt to sell endoscopes.
Nothing new here.”

119. Submitted to:
Nature
Nature Medicine
Science
Nature Communications
PLoS 1
PNAS
Journal of Pathology
Scientific Reports
Reviewer 3: I’d believe it if you showed it in mice…

121.  2,419 news articles that mentioned the study
 13,600+ original social media posts related
to the study (11,000+ on Twitter alone)
 672 million potential social media views

122.  2,419 news articles that mentioned the study
 13,600+ original social media posts related
to the study (11,000+ on Twitter alone)
 672 million potential social media views
Total estimated people reached:
3.8 billion!!!

123. Total estimated people reached:
3.8 billion!!!
… and then it got translated into Chinese…
 2,419 news articles that mentioned the study
 13,600+ original social media posts related
to the study (11,000+ on Twitter alone)
 672 million potential social media views

126. Next up!

127. Fluid, known:
 Rich in HA and proteoglycans => gel/syrup
BUT: FITC flows fast
>>> ? Filtration system ?
 Collaboration with Microfluidics lab at Penn
Engineering
 Small molecule contents with Michael Pacold
& Drew Jones, NYU Metabolomics Core
 Dye injection in pig bile ducts and livers at
Penn Vet
Matrix bundles
 Type 1/3 collagen bundles generate
piezo electricity
 Collaboration with Larry Miller at NorthWell
 Derivation: collagen-GFP from lab of Tatiana
Kisseleva and David Brenner (UCSD)
 EFEMP1 (fibulin-3) null mice to evaluate role
of elastin fibers (Nancy Spinner, Marcella
DeVoto at CHOP)
Interstitial lining cells:
 Mesenchymal stem cells?
 “Anti-tumoral fibroblasts”?
 Single cell sequencing in neonates and
adults with Katalin Susztak, Penn Renal

128. Fluid, known:
 Rich in HA and proteoglycans => gel/syrup
BUT: FITC flows fast
>>> ? Filtration system ?
 Collaboration with Microfluidics lab at Penn
Engineering
 Small molecule contents with Michael Pacold
& Drew Jones, NYU Metabolomics Core
 Dye injection in pig bile ducts and livers at
Penn Vet
Matrix bundles
 Type 1/3 collagen bundles generate
piezo electricity
 Collaboration with Larry Miller at NorthWell
 Derivation: collagen-GFP from lab of Tatiana
Kisseleva and David Brenner (UCSD)
 EFEMP1 (fibulin-3) null mice to evaluate role
of elastin fibers (Nancy Spinner, Marcella
DeVoto at CHOP)
Interstitial lining cells:
 Mesenchymal stem cells?
 “Anti-tumoral fibroblasts”?
 Single cell sequencing in neonates and
adults with Katalin Susztak, Penn Renal

129. Dept. of Medicine, Digestive Disease Division
Mount Sinai Beth Israel Medical Center
 Petros Benias, MD (now Northwell)
 David Carr-Locke, MD (now Cornell)
 Darren Buonocore
 Markus Miranda
Dept. of Pathology (Electron Microscopy Laboratory)
Mount Sinai Beth Israel Medical Center
 Jason Reidy, PhD
Dept. of Pathology, NYU School of Medicine
 Sue Kornacki, Stella Gordin, Syed Hoda
Rebecca Wells Laboratory,
Dept. of Medicine, Gastroenterology
University of Pennsylvania
 Bridget Sackey, PhD
 Jessica Llewelyn, PhD