A little exercise goes a long way to deliver chemotherapy

Keri SchadlerMay 21, 2015

McDonald D and Choyke P. Nature Medicine 2003

Normal Vasculature Tumor Vasculature

Tumor vasculature is inefficient: Drug Delivery is Ineficient

Tumor vasculature can be ‘normalized’

Jain R. Science 2005

Goel S. et al., Physiol. Rev. 2011

Tumor vascular normalization enhances drug delivery

Increased perfusion after anti-angiogenic therapy correlates with survival in glioblastoma patients

AG Sorenson, et al.. Cancer Research 2012 Jan 15;72(2):402-7.

Limitations in the use of anti-angiogenic therapy for vascular normalization

Increased shear stress activates the endothelium and remodels the vasculature

network

• Shear stress is regulated by the rate of blood flow and the vessel diameter

Shear Stress Regulates Vascular Remodeling

Disturbed Shear (Tumor)- High EC Turnover:

proliferation & apoptosis

- High Permeability- Immature Vessels- Sporadic Blood Flow

Moderate Steady Shear– Quiescent ECs– Low permeability– Mature Vessels– Constant blood flow

Nat Rev Mol Cell Biol. 2009 January; 10(1): 53–62

Exercise increases shear stress

Suh et al., Ann Biomed Eng. 2011

Aerobic exercise

Heart rate

blood flow

shear stress

Mea

n w

all s

hear

stre

ss

(dyn

es/c

m2 )

Exercise is safe and feasible for cancer patients

Can Exercise Normalize Tumor Blood Vessels?

Jain R. Science 2005

The models: Mice, Cancer, and Exercise

HighModerateLow

50% 70%

12m/min ~ Brisk Walk45 min/day; 5 days/week

Begin when tumor is palpable

VO2 :

B16F10 Melanoma or PDAC-4662 Pancreatic Adenocarcinoma4T1 or E0771 Mammary Carcinoma

Voluntary wheel runningBegan day of tumor inoculation

Exercise Normalizes Tumor VasculaturePDAC

No Ex Ex

CD31; DAPI

4T1 Mammary

Vessel Length

*Lumens

*

B16F10 Melanoma

Pericyte Coverage Increases with Exercise

ExerciseNo Exercise4T1 Mammary

Exercise Increases Tumor Vessel Function

No Ex Ex

4T1 Mammary Tumors

Exercise Increases Tumor Vessel Function

CD31 Lectin Merge; DAPI

No Ex

Ex

*

PDAC tumors, n=5-6 per group

Exercise Induces Functional Endothelial Tubes in Matrigel

Lectin; DAPI Lectin;CD31;DAPI

No Ex

Ex

*

Shear Stress

Endothelial Cells in

Matrigel

Inject subcu; wait one day

Exercise (or not) 7 days Harvest

MatrigelShear Stress

Endothelial Cells in

Matrigel

Inject subcu; wait one day

Exercise (or not) 7 days Harvest

Matrigel

Dongha Bhang, UPenn

= Tumor Growth

Chemotherapy Delivery

Hypothesis: Exercise increases chemotherapy delivery and efficacy

The models: Mice, Cancer, and Exercise

12m/min ~ Brisk Walk45 min/day; 5 days/week

Begin when tumor is palpableDoxorubicin or Gemcitabine 2 or 3x weekly

B16F10 Melanoma or PDAC-4662 Pancreatic Adenocarcinoma4T1 or E0771 Mammary Carcinoma

Voluntary wheel runningBegan day of tumor inoculationCyclophosphamide days 7,9,11

Exercise Increases Chemo Efficacy

PDAC B16F10

4T1 Mammary

Chemotherapy delivery is increased in tumors from exercised mice

gH2AX DAPI

Gem Gem + Ex

*

Melanoma Lysates*

Functional vessels are made by balance of pro- and anti-angiogenic factors

4T1 whole tumor RNA: VEGF

Pro: VEGF Anti: TSP-1

TSP-1 is necessary for increased chemotherapy efficacy by exercise

PDAC tumors; Tsp1f/f ;VE-cad Cre mice

Gem + ExerTSP-1

b-actin

Whole Tumors

TSP-1

b-actin

Endothelial Cells

Hrs Shear: 0 24

Is normalization necessary, or is acute exercise enough?

= Tumor Growth

Chemotherapy Delivery

Melanoma: One exercise session does not increase doxorubicin delivery

B16F10 tumors; Dox assessed by spectrophotometry

Acute ExChronic Ex

*

How do tumor vessels respond to acute exercise?

5 min- Run 60 min - Rest

RatsMatLyLu prostate tumors

Treadmill: 15m/min, 10* incline (60% max capacity)

Danielle J. McCullough et al. JNCI J Natl Cancer Inst 2014;106:dju036

Acute exercise increases tumor blood flow and decreases vascular resistance

Blood Flow Vascular Resistance

Danielle J. McCullough et al. JNCI J Natl Cancer Inst 2014;106:dju036

Rest

Exer.

Acute exercise increases tumor blood volume

Danielle J. McCullough et al. JNCI J Natl Cancer Inst 2014;106:dju036

Tumor Arterioles Constrict Differently than Normal Vessels

Norepinephrine Constriction Intraluminal Pressure Response

Exercise Enhances Chemotherapeutic Efficacy

• Exercise Normalizes Tumor Vasculature• Exercise Causes Acute Increases in Tumor

Blood Flow• Blood Flow Promotes Tumor Growth…

=Tumor Growth

Blood Delivery

Is exercise a good thing when there’s no chemotherapy?

?

Exercise has cancer type-specific effects on tumor growth

*

B16F10 MelanomaPDAC-4662 Pancreatic Adeno.

4T1 Mammary Tumors

Allison S. Betof et al. JNCI J Natl Cancer Inst 2015;107:djv040

Allison S. Betof et al. JNCI J Natl Cancer Inst 2015;107:djv040

Exercise increases apoptosis in 4T1 mammary tumors

Exercise is Multi-faceted

Immune Response

Oxidative Stress/ROS

Metabolism

Cytokines / Growth Factors

IL-6IGF1

Work to be done…

• Acute vs Chronic?• What kind of exercise? What intensity?• Molecular Regulators?• Which patients will get the most benefit?• Combination with other therapies?• Effect on Metastasis? Overall survival?

Thank you…Collaborators Wanted!

In vitro studies of shear stress using 3D-lumenized blood vessel models

Chris Chen & Pete Galie,U Penn Bioengineering

Endothelial Tube

collagen

Pump Media Into Device Collect Media

Serum from Exercised Mice Inhibits EC Sprouting

No Exercise Serum Exercise Serum

*

Chris Chen & Pete Galie,U Penn Bioengineering

Shear Stress Conditioned Media Inhibits EC proliferation and tube formation

No shear C.M. Shear C.M.

WT mouse lung Endothelial Cells (ECs)*p<0.05, n =3

In this complex, PECAM-1 is hypothesized to be the primary mechanotransducer, where it is directly stimulated by force to initiate signaling. VE-cadherin functions as an adapter that brings VEGFR2 into proximity with PECAM-1, where VEGFR2 is activated by a src family kinase. Activated VEGFR2 then recruits PI3-kinase and mediates activation of downstream pathways. Recent work has revealed additional functions of these proteins in vascular remodeling and function, and shed new light on their involvement in mechanotransduction.

PLC

nucleus

PIP2

DAG

IP3

Ca2+

Endoplasimic Reticulum

NFATCalcineurin

P

P

Angiogenesis genes

NFAT

NFAT

TSP-1

Shear stress induces NFAT nuclear translocation

No

Shea

rSh

ear

NFATc1 NFATc1; DAPI

8 minutes shear stress, avg 10 dyne/cm2

*

Shear Stress induces NFAT gene targets

Rnd1Cox2

**

Hours Shear Stress

Cyclosporin (NFAT inhbition) abrogates exercise effect in B16F10 tumors

NFAT inhibition in DSCR1 Tg mice abrogates the exercise effect on PDAC tumors

Calcineurin

NFATp p

NFAT

DSCR1

Inhibition of NFAT prevents vascular normalization

Thrombospondin-1 (TSP-1): an NFAT regulated anti-angiogenic factor

TSP-1• Soluble, secreted factor• Produced by ECs,

stroma, immune• Receptor on ECs• Inhibits EC proliferation• Vessel Maturation

NFAT

IL-6Cox2Rnd1Ang2TSP-1

CD31; DAPI

CD31; DAPI

Tumor Growth

Chemotherapy Delivery

Inefficient Vasculature

“Normalized” Vasculature

Moderate Exercise

Shear Stressnucleus

Ca2

+

P

P

NFAT

TSP-1

NFAT

NFAT

Effects of aerobic exercise on angiogenesis and vascular maturity in primary 4T1 breast tumors.

Allison S. Betof et al. JNCI J Natl Cancer Inst 2015;107:djv040

© The Author 2015. Published by Oxford University Press.

Pharmacologic Increase in Shear Stress Also Enhances Gemcitabine Efficacy

*

Praz = Prazosin; PDAC tumors; 4-5 per group

p<0.05

Exercise inhibits Ewing’s sarcoma growth in mice

TC71 cells in nude mice, n = 3 -5 per group, Doxo = 4mg/kg/week

TSP-1 is needed for growth inhibition by shear stress conditioned media

Naïve WT cells

WT or Tsp1-/- ECconditioned media

Measure proliferation

Fluorescence photomicrograph of a prostate tumor cross-section, obtained from a MatLyLu Copenhagen rat prostate carcinoma after intraperitoneal injection of EF5 (30mg/kg) at rest

(A) and during acute exercise (B).

Danielle J. McCullough et al. JNCI J Natl Cancer Inst 2014;106:dju036

© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.