Harvard-MIT Division of Health Sciences and Technology
HST.525J: Tumor Pathophysiology and Transport Phenomena, Fall 2005
Course Director: Dr. Rakesh Jain
Delivery of Molecular and Cellular Medicine to Tumors
Vascular Transport
(Lecture I)
Angiogenesis &
Microcirculation
(Lecture I)
Interstitial and Lymphatic Transport
(Lecture II)
Vascular
Normalization
(Lecture Il)
1
DELIVERY OF MOLECULAR MEDICINE
TO TUMORS
Lecture II: Interstitial and Lymphatic Transport
OVERVIEW
R.K. Jain, “Transport of Macromolecules in the Tumor Interstitium: A Review,” Cancer Research, 3038- 47:3050 (1987).
R.K. Jain, “1996 Landis Award Lecture: Delivery of Molecular and Cellular Medicine to Solid Tumors,” Microcirculation,
4:1-23 (1997).
R.K. Jain, and B.T. Fenton, “Intra-Tumoral Lymphatic Vessels: A Case of Mistaken Identity or Malfunction?” JNCI,
94:417-421 (2002).
R.K. Jain, and T.P. Padera, “Prevention and Treatment of Lymphatic Metastasis by Anti-Lymphangiogenic Therapy,”
JNCI, 94:785-787 (2002).
Jussila L, Alitalo K, “Vascular growth factors and lymphangiogenesis,” Physiology Reviews, 82:673-700 (2002).
R.K. Jain and T. P. Padera, “ Lymphatics Make the Break,” Science. 299: 209-210 (2003)
R.K. Jain, “Molecular Regulation of Vessel Maturation,” Nature Medicine, 9:685-693 (2003).
R.K. Jain “Vascular and Interstitial Biology of Tumors,” In: Clinical Oncology, 3rd Edition (Editors: M. Abeleff, J.
Armitage, J. Niederhuber, M. Kastan and G. McKenna), Elsevier, Philadelphia, PA, Chapter 9, pp. 153-172 (2004).
2005
Interstitial Transport
S.R. Chary and R.K. Jain, “Direct Measurement of Interstitial Diffusion and Convection of Albumin in Normal and Neoplastic
Tissues Using Fluorescence Photo-bleaching,” PNAS, 86:5385-5389 (1989).
D.A. Berk, F. Yuan, M. Leunig and R.K. Jain, “Direct in vivo Measurement of Targeted Binding in a Human Tumor
Xenograft, “ PNAS, 94:1785-1790 (1997).
A. Pluen, P.A. Netti, R.K. Jain, and D.A. Berk, “Diffusion of Macromolecules in Agarose Gels: Comparison of Linear and
Globular Configurations,” Biophysical Journal, 77: 542-552 (1999).
P.A. Netti, D.A. Berk, M.A. Swartz, A.J. Grodzinsky and R.K. Jain, “Role of Extracellular Matrix Assembly in Interstitial
Transport in Solid Tumors,” Cancer Research, 60: 2497-2503 (2000).
A. Pluen, Y. Boucher, S. Ramanujan, T.D. McKee, T. Gohongi, E. di Tomaso, E.B. Brown, Y. Izumi, R.B. Campbell, D.A.
Berk, R.K. Jain, "Role of Tumor-Host Interactions in Interstitial Diffusion of Macromolecules: Cranial vs. Subcutaneous
tumors," PNAS, 98: 4628-4633 (2001).
C. de L. Davies, D.A. Berk, A. Pluen, and R.K. Jain, “Comparison of IgG Diffusion and Extracellular Matrix Composition in
Rhabdomyosarcomas Grown in Mice Versus In Vitro as Spheroids Reveals the Role of Host Stromal Cells,” BJC, 86:16391644 (2002)
S. Ramanujan, A. Pluen, R.D. McKee, E.B. Brown, Y. Boucher, R.K. Jain, “Diffusion and Convection in Collagen Gels:
Implications for Transport in the Tumor Interstitium,” Biophysical Journal. 83: 1650-1660 (2002).
E. Brown, T. McKee, E. di Tomaso, B. Seed, Y. Boucher, R.K. Jain, “Dynamic Imaging of Collagen and its Modulation in
Tumors in vivo using Second Harmonic Generation,”Nature Medicine. 9: 796-801 (2003).
G. Alexandrakis, E. B. Brown, R. T. Tong, T. D. McKee, R. B. Campbell, Y. Boucher and R. K. Jain, “Two-photon
fluorescence correlation microscopy reveals the two-phase nature of transport in tumors,” Nature Medicine, 10:203-207
(2004).
E. B. Brown, Y. Boucher, S. Nasser, and R. K. Jain,”Measurement of Macromolecular Diffusion Coefficients in Human
Tumors”, Microvascular Research, 67 231-236 (2004).
3
2005
Lymphatic Transport
A. Leu, D.A. Berk, F. Yuan and R.K. Jain, “Flow Velocity in the Superficial Lymphatic Network of the Mouse Tail,”
American Journal of Physiology, 267:H1507-H1513 (1994).
M.A. Swartz, D.A. Berk and R.K. Jain, “Transport in Lymphatic Capillaries: I. Macroscopic Measurements Using
Residence Time Distribution Theory,” American Journal of Physiology, 270:H324-H329 (1996).
D.A. Berk, M.A. Swartz, A.J. Leu, and R.K. Jain, “Transport in Lymphatic Capillaries: II. Microscopic Velocity
Measurement with Fluorescence Recovery After Photobleaching,” American Journal of Physiology, 270:H330-H337
(1996).
M. Jeltsch, A. Kaipainen, M. Swartz, D. Fukumura, R.K. Jain, and K. Alitalo, “Hyperplasia of Lymphatic Vessels in VEGFC Transgenic Mice,” Science, 276:1423-1425 (1997).
S.A. Slavin, A.D. van den Abbeele, A. Losken, M. Swartz, and R.K. Jain, “Return of Lymphatic Function Following Flap
Transfer for Acute Lymphedema,” Annals of Surgery, 229:421-427 (1999).
M.A. Swartz, A. Kaipainen, P. Netti, C. Brekken, Y. Boucher, A.J.Grodzinsky and R.K.Jain, “Mechanics of InterstitialLymphatic Fluid Transport: Theoretical Foundation and Experimental Validation,” Journal of Biomechanics, 32: 1297-1303
(1999).
A.J. Leu, D.A. Berk, A. Lymboussaki, K. Alitalo and R.K. Jain, “Absence of Functional Lymphatics within a Murine
Sarcoma: a Molecular and Functional Evaluation,” Cancer Research, 60: 4324-4327 (2000).
M.A. Swartz, A. Kaipainen, P. Netti, C. Brekken, Y. Boucher, A.J.Grodzinsky and R.K.Jain, “Mechanics of InterstitialLymphatic Fluid Transport: Theoretical Foundation and Experimental Validation,” Journal of Biomechanics, 32: 1297-1303
(1999).
A.J. Leu, D.A. Berk, A. Lymboussaki, K. Alitalo and R.K. Jain, “Absence of Functional Lymphatics within a Murine
Sarcoma: a Molecular and Functional Evaluation,” Cancer Research, 60: 4324-4327 (2000).
A. Kadambi, C.M. Carreira, C.-O. Yun, T.P. Padera, D.E.J.G.J. Dolmans, P. Carmeliet, D. Fukumura and R.K. Jain.
“Vascular endothelial Growth Factor (VEGF)-C Differentially Affects Tumor Vascular Function and Leukocyte
Recruitment: Role of VEGF-Receptor 2 and Host VEGF-A,” Cancer Research, 61: 2404-2408 (2001).
4
2005
C. Mouta Carreira, S.M. Nasse, E. diTomaso, T.P. Padera, Y. Boucher, S.I. Tomarev, R.K. Jain, “LYVE-1 is not
Restricted to the Lymph Vessels: Expression in Normal Liver Blood Sinusoids and Down-regulation in Human Liver
Cancer and Cirrhosis,” Cancer Research, 61:8079-8084 (2001).
T.P. Padera, B.R. Stoll, P.T.C. So, and R.K. Jain, “High-Speed Intravital Multiphoton Laser Scanning Microscopy of
Microvasculature, Lymphatics, and Leukocyte-Endothelial Interactions,” Molecular Imaging, 1:9-15 (2002)
T.P. Padera, E. di Tomaso, A. Kadambi, C. Mouta Carreira, E.B. Brown, Y. Boucher, N.C. Choi, D. Mathisen, J. Wain,
E.J. Mark, L.L. Munn, R.K. Jain, “Lymphatic Metastasis in the Absence of Functional Intratumor Lymphatics,” Science,
296:1883-1886 (2002)
T.P. Padera, E. di Tomaso, A. Kadambi, C. Mouta Carreira, E.B. Brown, Y. Boucher, N.C. Choi, D. Mathisen, J. Wain,
E.J. Mark, L.L. Munn, R.K. Jain, “Lymphatic Metastasis in the Absence of Functional Intratumor Lymphatics,” Science,
296:1883-1886 (2002)
T. Padera, B. Stoll, J. Tooredman, D. Capen, E. di Tomaso, and R. K. Jain, “Cancer cells compress intratumor vessels,”
Nature, 427: 695 (2004).
J. Hagendoorn, T.P. Padera, S. Kashiwagi, N. Isaka, F. Noda, M.I. Lin, P.L.Huang, W.C. Sessa, D. Fukumura, and R.K.
Jain. “Endothelial nitric oxide synthase regulates microlymphatic flow via collecting lymphatics,” Circulation Research, 95:
204-9 (2004).
N. Isaka, T. P. Padera, J. Hagendoorn, D. Fukumura, and R. K. Jain, “Peritumor lymphatics induced by vascular
endothelial growth factor-C exhibit abnormal function,” Cancer Research, 64: 4400-4404 (2004).
5
2005
Interstitial Transport
Figure by MIT OCW.
6
2005
Measuring Convection, Diffusion and Binding
7
2005
Fluorescence Recovery After Photobleaching
I0
I/I0
0
8
time
5
2005
Typical Values of Interstitial Convective Velocities &
Directions
9
Reference: Chary & Jain, PNAS (1989)
2005
Interstitial Diffusion of IgG in LS174T Xenograft
Dmean = 1.3 x10-7
50
Count
40
30
D0
20
10
0
10-8
10-7.5
10-7
10-6.5
10-6
2
D (cm /s)
1
0.8
0.6
D/D0
0.4
0.2
0
10
Fab'
Albumin
IgG
Diffusing molecule
Reference: Berk et al. PNAS (1997)
2005
Time Constant for Diffusion
11
Distance
IgG
Fab
100 µm
~ 0.5 hours
~ 0.2 hours
1 mm
~ 2–3 days
~ 0.5–1 days
1 cm
~ 7 months
~ 2 months
Plasma
Half-life
~ days
~ hours
2005
Specific Antibody
1 sec
10 sec
100 sec
Non-Specific Antibody
12
Reference: Berk et al. PNAS (1997)
2005
Effect of Molecular Configuration on Diffusion
10
-5
10
-6
2
s
-1
in 0.1M PBS
Diffusion
coefficient
<r >
p
10
-7
10
-8
10
-9
10
<rp>: experimental
pore radius
"Spherical"
molecules
Molecular size
DNA
-10
1
10
100
Experimental Hydrodynamic Radius, R
1000
H
, nm
13
Reference: Pluen et al. Biophysical Journal (1999)
Interstitial diffusion varies with site of growth
Dorsal window
Diffusion coefficient (cm2s-1)
2005
Cranial window
10-5
10-6
10-7
10-8
10-9
10-10
0.1
14
1
10
100
Hydrodynamic radius (nm)
Courtesy of National Academy of Sciences, U. S. A. Used with permission.
Source: Tomaso, E., E. B. Brown, Y. Izumi, R. B. Campbell, D. A. Berk, R. K. Jain. "Role of tumor-host interactions in interstitial diffusion of macromolecules:
cranial vs. subc Pluen A, Boucher Y, Ramanujan S, McKee TD, Gohongi T, di utaneous tumors." Proc Natl Acad Sci 98 (2001): 4628-4633. (c) National
Academy of Sciences, U.S.A.
2005
Tumor-Host Interaction Influences Drug Transport
Image removed for copyright reasons.
Multicell spheroid human squamous cell carcinoma.
See: Science 240, no. 4849 (April 8, 1988): cover page.
15
Figure by MIT OCW. After Jain.
2005
Role of Collagen in Interstitial Transport
15
10
Collagen
content
(mg/g)
5
0
MCa-IV
LS174T
HSTS
U87
Tumor
Increase
in D (%)
16
120
100
80
60
40
20
0
-20
Treatment:
saline
collagenase
(N=3)
(N=4)
Reference: Netti et al. Cancer Research (2000)
2005
Collagen Restricts HSV Distribution
50 µm
Fibrillar collagen
HSV particles (150 nm)
17
Reference: McKee, Grandi, Mok et al., submitted (2005)
2005
Collagenase Co-Injection Improves Virus Distribution
1 mm
Virus Alone
1 mm
18
Collagenase Co-injection
Reference: McKee, Grandi, Mok et al., submitted (2005)
2005
Collagenase Improves Efficacy of MGH2
Oncolytic Viral Therapy
Courtesy of the American Association for Cancer Research. Used with permission. From McKee, T. D., P. Grandi, W. Mok, G. Alexandrakis,
N. Insin, J. P. Zimmer, M. G. Bawendi, Y. Boucher, X. O. Breakefield, and R. K. Jain. "Degradation of fibrillar collagen in a human
melanoma xenograft improves the efficacy of an oncolytic herpes simplex virus vector." Cancer Research 66 (2006): 2509-2513.
19
2005
Relaxin
fibroblasts
Decreases
collagen
synthesis
20
Decreased
collagen production
Increases
MMPs
Decreases
TIMPs
Increased collagen degradation
2005
Relaxin treatment modifies collagen structure
Day 0
Day 2
Day 5
Day 8
Day 9
Day 12
75 µm
relaxin
21
placebo
Reference: Brown, McKee et al., Nature Medicine (2003)
2005
Relaxin treatment enhances macromolecular transport
2 -1
Diffusion coefficients, D, cm s
4
3.5 10
-7
3.0 10
-7
2.5 10
-7
2.0 10
-7
1.5 10
-7
1.0 10
-7
5.0 10
-8
3.5
3
2.5
2
1.5
1
0.5
7.0 10
-9
6.0 10
-9
5.0 10
-9
4.0 10
-9
3.0 10
-9
2.0 10
-9
1.0 10
-9
0
w/o Relaxin
1
22
IgG
w Relaxin
1.5
w/o Relaxin
2
w Relaxin
2.5
Dextran 2,000,000 MW
Reference: Brown, McKee et al. Nature Medicine (2003)
2005
Summary
• FRAP can provide direct measurements of diffusion, convection
and binding in vivo
• Convection
~ 0.1 - µm/sec
• Diffusion
~ 1/3 of water
• Non-specific binding
~ 1/4 - 1/3
• Interfering with collagen synthesis and/or assembly may
enhance interstitial transport.
• Interstitial diffusion depends on the host-tumor interaction.
• Fluorescence correlation microscopy can reveal the two-phase
nature of transport in tumors.
23
2005
25
Courtesy of Lance Munn. Used with permission.
2005
Intravital Lymphangiography
26
Reference: Leu et al. Am J Physiol. (1994)
Hagendoorn et al. (2004)
2005
27
2005
Lack of Functional Lymphatics in Tumors
Normal tail
Tumor bearing
Courtesy of the American Association for Cancer Research. Used with permission. From Leu, A. J., D. A. Berk, A. Lymboussaki,
K. Alitalo, and R. K. Jain. "Absence of functional lymphatics within a murine sarcoma: a molecular and functional evaluation."
Cancer Research 60 (2000): 4324-4327.
28
2005
VEGF-C Induces Lymphatic Hyperplasia
Control – Lymph Vessels
VEGF-C Induced Hyperplasia
29
Reference: Jeltsch et al. Science, (1997)
2005
Effect of VEGF-C on Lymphatic Metastases
Property
P-value*
Cell Line
B16F10-MT B16F10-VEGF-C
Lymphatic Metastasis 6/21
15/19
Local Invasion
3/21
12/18
5/22
2/19
Lung Metastasis†
0.002
0.001
N.S.
T-241-MT
Lymphatic Metastasis 2/14
Local Invasion
4/14
†
Lung Metastasis
5/14
0.046
N.S.
N.S.
T-241-VEGF-C
8/14
5/14
6/14
Numerator is the number of mice that exhibited the property, denominator is
the total number of mice.
*: P-value based on Fisher’s Exact Test. N.S.: not significant
†: Hematogenous metastasis was analyzed microscopically in 10-20 sections
of lung per animal.
30
Reference: Padera et al., Science, (2002)
2005
Lymphatic Function Studies
• Interstitial fluid pressure measurements
• Epifluorescence lymphangiography
• Multi-photon laser scanning microscopy
• Ferritin lymphangiography
Immunohistochemistry
• Lectin, CD31, LYVE-1, Prox 1
31
2005
IFP is not changed by VEGF-C overexpression
25
*
20
*
15
10
5
0
Normal tissue
T-241-MT
T-241-VC+
p < 0.05
* compared
to normal
tissue
32
Reference: Padera et al., Science, (2002)
2005
Diameter (microns)
VEGF-C induced hyperplasia of peri-tumor lymphatics
60
50
40
30
20
10
0
MT
VEGF-C
Green: Lymphatic vessels
Red: Blood vessels
33
Reference: Padera et al., Science, (2002)
2005
HCCs in Patients Lack Lymphatics
Lymphatic vessels
Peritumor
Tumor
•42 biopsies of liver tumor (HCC + metastases) examined
•No staining for Prox 1 + LYVE-1 was observed in the tumor area
Courtesy of the American Association for Cancer Research. Used with permission. From C, Mouta Carreira, S. M. Nasser, E. di Tomaso,
T. P. Padera, Y. Boucher, S. I. Tomarev, and R. K. Jain. "LYVE-1 is not restricted to the lymph vessels: expression in normal liver blood
sinusoids and down-regulation in human liver cancer and cirrhosis." Cancer Research 61 (2001): 8079-8084.
34
2005
Human Lung Tumors
Lymphatic
vessels
Tumor edge
Lymphatic
vessels
875 microns
200 microns
IFP = 9.5 ± 1.5 mmHg
n = 27
35
Reference: Padera et al., Science (2002)
2005
Fluid pressure can compress
impermeable objects
Fluid pressure can NOT compress
permeable vessels, such as blood and
lymphatic vessels
Solid stress can compress
permeable vessels, such as
blood and lymphatic vessels
36
Courtesy of Lance Munn. Used with permission.
2005
DT treatment opens lymphatic vessels
37
Reference: Padera, Stoll et al. Nature, (2004)
Peritumor lymphatics induced by VEGF-C
exhibit abnormal function
2005
Normal tissue
B16F10-MT
B16F10-VEGF-C
T
Lymphatics
T
Retrograde lymphatics
*
38
10
10
5
5
0
0
Control
MT VEGF-C
*
Control
MT
VEGF-C
Courtesy of the American Association for Cancer Research. Used with permission. From Isaka, N., T. P. Padera, J. Hagendoorn, D. Fukumura, and R. K. Jain.
"Peritumor lymphatics induced by vascular endothelial growth factor-C exhibit abnormal function." Cancer Research 64 (2004): 4400-4404.
2005
Image removed for copyright reasons.
See: Jain, R.K., and B.T. Fenton. "Intra-Tumoral Lymphatic Vessels: A Case of Mistaken Identity or Malfunction?"
JNCI 94 (2002): 417-421.
39
2005
Summary
• Despite the presence of VEGF-C and its receptor VEGFR3,
functional lymphatics cannot be detected in tumors.
• VEGF-C overexpression increases diameter and number of peritumor lymphatics.
• Overexpression of VEGF-C and -D increases vascular
angiogenesis.
• Levels of VEGF-C and -D correlate with increased lymphatic and
hematogeneous metastasis.
• Blocking VEGF-C/-D can prevent lymphatic metastasis.
40
2005
Overall Summary
Mechanisms of Heterogeneous Distribution of
Macromolecules in Tumors
• Heterogeneous blood flow
• Elevated interstitial pressure
• Fluid oozing from the periphery
• Slow interstitial diffusion
• Retardation by binding
• Heterogeneous expression of antigens
41
2005
Strategies for Improved Delivery
• Make the vasculature the target for therapy
• Modify physiological parameters
– Increase perfusion
– Modulate interstitial pressure
– Interfere with collagen synthesis (e.g., relaxin)
– Use multi-step strategies
– “Normalize” the abnormal tumor vasculature
42