1. Tumors Models, and Response of Tumors Martin Brown April 23, 2012

2. Cancers comprise more than tumor cells
Cancers comprise more than tumor cells. Do the stromal cells affect the response to radiotherapy?
Hanahan and Weinberg, Cell 2011

3. Endothelial cells should be a prime target in radiotherapy – Julie Denekamp
Capillary
Endothelial cell
Endothelial cells in tumors are rapidly dividing so will die rapidly after irradiation
One endothelial cell supports 2000 tumor cells!

4. Tumor curability by IR does not depend on the sensitivity of the host (Budach et al, 1993)

6. TD50 assay for in vivo response of tumors
(Hewitt and
Wilson, 1959)

7. Cell survival curve of leukemia cells irradiated in situ and in vitro (Hewitt & Wilson,1959)

8. Lung colony assay (Hill and Bush)

9. Tumor Growth Delay Assay

10. Tumor growth delay following different radiation doses.

11. Demonstration of Tumor Radiosensitization using TCD50 assay (Sheldon et al, 1974)

12. In vivo/in vitro (excision) assay
This is the fastest, cheapest and most accurate assay of the response
of the tumor cells. However, it makes the assumption that the response
of the cells is not altered by taking them out of the tumor.

13. Demonstration of “hypoxic fraction” in mouse tumors (Rockwell & Kallman, 1972)

14. Spheroids: an in vitro tumor model.
Mimics several aspects of tumors
-chronic hypoxia adjacent to necrosis
-non dividing cells
-cell to cell contact
-limited diffusion of drugs

15. Tumor Hypoxia results from Differences in the Vasculature between Tumor and Normal Tissues

16. Chronic Hypoxia (HIF-1 staining) in Human Head & Neck Cancers(Aebersold et al, 2001)
Blood vessels
Necrosis

17. Oxygenation of human tumors is usually measured with an oxygen electrode (“Eppendorf”)

18. Oxygen tensions of Normal Tissue and Nodes of Head and Neck Cancers20 16
Normal Subcutaneous Tissue 12 8 4
Median pO2 10 20 30 40 50 60 70 80 90
100
% of Measurements 20
Tumor 16 12 8 4 10 20 30 40 50 60 70 80 90
100
Oxygen Partial Pressure (mm Hg)

19. Median pO2 Values for Tumor and Normal
Tissues for 62 Head & Neck Cancer Patients (Le et al 2004)
Median pO2 values for Tumor and Normal
0.3
Tumor Median
= 11.8 mmHg
Normal Median
= 51.9 mmHg
0.2
Proportion of
Values
0.1
0.0 10 20 30 40 50 60 70 80
Median pO2 (mmHg)

20. Effect of Tumor Hypoxia on Local Control of Head and Neck Cancer (Brizel et al, 1999)
63 patients treated.
Similar differences
for survival and DFS.

21. Oxygenation of Prostate Carcinoma by Eppendorf Electrode
Movsas et al, 2001
Parker et al, 2004
Med = 2.4mmHg
Med = 4.5 mmHg

22. Effect of tumor hypoxia on outcome of prostate ca to RT (Movsas et al, 2002)
pO2 = 5.3 mmHg
pO2 = 1.0 mmHg
Biochemical failure by PSA

23. Median Oxygen Levels of Human Tumors
(Brown & Wilson Nat. Rev. Cancer 4: )

24. It is incorrect to think that tumors are composed of hypoxic and aerobic cells. In fact the majority are at intermediate pO2
Hypoxic
Aerobic
Intermediate
0.1 1 10
100
1.0
1.5
2.0
2.5
3.0
OER(p)
Oxygen Partial Pressure (p) (mm Hg)
Koch et al,1984
Whillans and
Hunt,1982

25. When the cells at intermediate hypoxia are considered the predicted survival to 2 Gy fractions is dramatically altered 10
10% hypoxic cells with full reoxygenation (binary)
10% hypoxic cells with no reoxygenation
10% hypoxic cells and intermediate p02 cells with full reoxygenation -1 -2 -3 -4
Surviving Fraction -5 -6 -7 -8
No Hypoxic Cells -9
-10 10 20 30 40 50 60
Total Dose (2 Gy Fractions)
Wouters and Brown,1997

26. Consequences of Tumor Hypoxia for Cancer Treatment
Hypoxic cells are resistant to killing by IR
Extent of hypoxia affects response to radiotherapy
Hypoxia is associated with slowing of proliferation:
Leads to resistance to most anticancer drugs.
Hypoxia promotes and selects for a more malignant phenotype
Hypoxic tumors are more metastatic

27. Hypoxia and proliferation in human H & N tumors (Van der Kogel et al)
Green: hypoxia
Red: proliferation (IdUrd+)

28. Human Glioblastoma Stained with EF5 (Red) and Ki-67 (Green) (Evans and Koch, 2002)

29. Expect gradient of cell killing from blood vessels to necrosis
Proliferation
Capillary
Surviving Fraction O2 O2 O2
Radiation / Chem. Drug .
Drug concentration 50
100
150
Distance from Capillary (µm)

30. X-ray Survival of Cells in SiHa Tumors as a function of Distance from Blood Vessels
(Durand &
Olive, 1997)
Aerobic
Hypoxic

31. Expression of hypoxia induced proteins (eg CA IX) can be used in archival tissues as a surrogate for hypoxia.
Immunohistochemical staining for CA IX
Head and Neck
NPC
Breast
Adenocarcinoma
Ovarian
Adenocarcinoma
Hui et al, 2002
Wykoff et al, 2000

32. Can we exploit tumor hypoxia?
A drug specifically toxic to hypoxic cells would kill only tumor cells - and the most resistant ones.

33. Preferential Toxicity of Tirapazamine (TPZ) to Hypoxic Cells in vitro2 O N NH
TPZ
Mouse SCCVII cells 1 hr exposure 1 2 3 4 - 5 T P Z C o n c ( M )
HCR=300
Surviving Fraction
Mouse SCCVII cells 1 hr exposure 1 2 3 4 - 5 T P Z C o n c ( M )
Hypoxia
Air

34. Proliferation Drug concentration
Addition of a Hypoxic Cytotoxin to Standard
Treatment Could Exploit Tumor Hypoxia
Hypoxic Cytotoxin
Proliferation
Capillary
Surviving Fraction O2 O2 O2
Radiation / Chem. Drug .
Combined
Drug concentration 50
100
150
Distance from Capillary (µm)

35. Complementary Killing by TPZ and IR in SiHa Tumors as a function of Distance from Blood Vessels
(Durand &
Olive, 1997)
Combined
Aerobic
Hypoxic

36. Primary site failure in 92 randomized advanced H&N patients (Peters et al,2007,)
PET hypoxia status
Treatment
P-value
RT + cis /no TPZ
RT + cis/TPZ
Non-Hypoxic
2/27 (7%)
3/21 (14%) NS
Hypoxic
8/18 (44%)
0/26 (0%)
0.0002
0.008

37. Results of multicenter randomized clinical trial of TPZ with advanced H & N cancer
All 863 patients in 89 sites in 16 countries
693 patients without major protocol violations
Need to select hypoxic tumors in future trials and have good QA for radiotherapy
Rischin et al and Peters et al, JCO, 2010

38. Patient Numbers Needed to Detect a Change from 40 to 65% Response Rates
10000
90% probability
80% probability
Number of
Patients
1000
100
100 80 60 40 20
% Patients with Hypoxic Tumors

39. SN30000 is superior to TPZ in multiple tumor models
Single doses ( 15 or 20 Gy)
Fractionated 8 x 2 Gy SiHa tumor
Hicks et al, Clin Cancer Res 2010

40. Summary
Sensitivity of tumor and normal cells can be assayed quantitatively in situ in experimental systems.
Tumor hypoxia has a major negative impact on the curability of tumors by radiotherapy, and probably also chemotherapy
Tumor hypoxia can be exploited using drugs specifically toxic to hypoxic cells.