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

Tumors Models, and Response of Tumors Martin Brown April 23, 2012. Cancers comprise more than tumor cells. Do the stromal cells affect the response to radiotherapy?. Hanahan and Weinberg, Cell 2011. Endothelial cells should be a prime target in radiotherapy – Julie Denekamp. Capillary.

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

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  1. Tumors Models, and Response of Tumors Martin BrownApril 23, 2012

  2. 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 cells in tumors are rapidly dividing so will die rapidly after irradiation • One endothelial cell supports 2000 tumor cells! Endothelial cell

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

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

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

  7. Lung colony assay (Hill and Bush)

  8. Tumor Growth Delay Assay

  9. Tumor growth delay following different radiation doses.

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

  11. 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.

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

  13. 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

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

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

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

  17. 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90 100 Oxygen tensions of Normal Tissue and Nodes of Head and Neck Cancers 20 Normal Subcutaneous Tissue 16 12 8 4 Median pO2 0 0 100 % of Measurements 20 Tumor 16 12 8 4 0 Oxygen Partial Pressure (mm Hg)

  18. Tumor Median = 11.8 mmHg Normal Median = 51.9 mmHg 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 0.2 Proportion of Values 0.1 0.0 0 20 30 40 50 60 70 80 10 Median pO2 (mmHg)

  19. 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.

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

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

  22. Median Oxygen Levels of Human Tumors (Brown & Wilson Nat. Rev. Cancer 4: 437 2004)

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

  24. 10 10 10% hypoxic cells with no reoxygenation 10 10 10 10% hypoxic cells and intermediate p02 cells with full reoxygenation 10 10 10 10% hypoxic cells with full reoxygenation (binary) 10 10 10 When the cells at intermediate hypoxia are considered the predicted survival to 2 Gy fractions is dramatically altered 0 -1 -2 -3 -4 Surviving Fraction -5 -6 -7 -8 No Hypoxic Cells -9 -10 0 10 20 30 40 50 60 Total Dose (2 Gy Fractions) Wouters and Brown,1997

  25. 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

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

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

  28. O2 Expect gradient of cell killing from blood vessels to necrosis Proliferation Capillary Surviving Fraction O2 O2 Radiation / Chem. Drug . Drug concentration 0 50 100 150 Distance from Capillary (µm)

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

  30. 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

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

  32. Mouse SCCVII cells 1 hr exposure 1 0 0 1 0 0 1 0 - 1 1 0 - 1 HCR=300 1 0 - 2 - 2 1 0 Surviving Fraction 1 0 - 3 1 0 - 3 Hypoxia 1 0 - 4 1 0 - 4 Air 1 0 - 5 1 0 - 5 0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0 0 1 2 3 4 1 0 1 0 1 0 1 0 1 0 T P Z C o n c ( µ M ) T P Z C o n c ( µ M ) Preferential Toxicity of Tirapazamine (TPZ) to Hypoxic Cells in vitro O N N Mouse SCCVII cells 1 hr exposure NH N 2 O TPZ

  33. Hypoxic Cytotoxin Combined O2 Addition of a Hypoxic Cytotoxin to Standard Treatment Could Exploit Tumor Hypoxia Proliferation Capillary Surviving Fraction O2 O2 Radiation / Chem. Drug . Drug concentration 0 50 100 150 Distance from Capillary (µm)

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

  35. Primary site failure in 92 randomized advanced H&N patients (Peters et al,2007,)

  36. 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

  37. 90% probability 80% probability 0 20 40 60 80 100 Patient Numbers Needed to Detect a Change from 40 to 65% Response Rates 10000 Number of Patients 1000 100 % Patients with Hypoxic Tumors

  38. 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

  39. 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.

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