METHOD FOR DETERMINING THE LEVEL OF HYPOXIA IN A TUMOR

Abstract

The invention is in the field of medicine and molecular therapeutics. The invention provides means and measures for diagnosis and treatment, in particular for diagnosis and treatment of tumors, more in particular for determining the level of hypoxia in a tumor and for reducing the risk of metastasis of a tumor in a subject. The invention comprises a method wherein a level of exosome-associated GABARAPL1 is determined in a bodily fluid of the subject and wherein an elevated level of exosome-associated GABARAPL1 is indicative of an increased level of hypoxia of the tumor. The invention also comprises an antibody directed against GABARAPL1 for use in the treatment or prevention of cancer.

Claims

1. A method for determining a level of hypoxia in a tumor of exosome-associated GABARAPL1 in a subject having a tumor, the method comprising: a determining the level of exosome-associated GABARAPL1 in a bodily fluid of the subject.

2. The method according to claim 1, wherein the bodily fluid is blood, serum or plasma.

3. The method according to claim 1 further comprising isolating an exosome and determining the level of exosome-associated GABARAPL1.

4. The method according to claim 1, wherein the tumor is a solid tumor.

5. The method according to claim 1, wherein the level of exosome-associated GABARAPL1 is determined using a method selected from the group consisting of immunoblotting, ELISA, and flow cytometry.

6. A method of treating a subject having a tumor, the method comprising: administering to the subject an antibody directed against GABARAPL1.

7. The method according to claim 6, wherein the risk of metastasis of the tumor in the subject is reduced.

8. The method according to claim 6, wherein GABARAPL1-containing exosomes are removed from circulation.

9. The method according to claim 6, wherein the antibody is administered intravascularly.

10. The method according to claim 6, wherein angiogenesis or neovascularization of the tumor is inhibited, decreased, or prevented.

11. The method according to claim 6, wherein the tumor is a solid tumor.

12. The method according to claim 1, further comprising comparing the determined level of exosome-associated GABARAPL1 to a control level.

Description

LEGEND TO THE FIGURES

[0045] FIG. 1: (A) Tumor growth, (B) vessel density and (C) hypoxic fraction of control (shSCR) and GABARAPL1 knockdown (shGABARAPL1) tumors.

[0046] FIG. 2: (A) tube formation by HUVEC after exposure to exosomes derived from control (SCR) or GABARAPL1 deficient tumour cells exposed to hypoxia. (B) inhibition of hypoxia-associated-exosome tube formation by anti-GABARAPL1 antibody. (C) anti-GABARAPL1 is only capable of reducing tube formation when initiated by exposure to exosomes.

[0047] FIG. 3: MDA-MB-231 adherence to HUVEC monolayers. HUVEC pre-exposure to exosomes facilitates adherence of tumor cells. This effect can be abrogated by GABARAPL1 blocking antibodies.

[0048] FIG. 4: MDA-MB-231 lung metastases. Control and GABARAPL1 knockdown cells were implanted orthotopically in nude mice. After reaching 1500 mm3, the lungs were excised and the number of metastasis were assessed.

[0049] FIG. 5: Correlation of plasma GABARAPL1 exosomes with HX4 high volume in NSCLC patients.

EXAMPLES

Example 1: Cell Culturing

[0050] MCF7 (mammary adenocarcinoma), HT29 (colorectal adenocarcinoma), and u87 (glioblastoma) cell lines were cultured in RPMI and DMEM (GE healthcare) growth media respectively with 10% FCS in a 5% CO2 incubator at 37 C. For hypoxia exposure, cells were transferred to ananoxic (0.0% O2) culture chamber (MACS VA500 microaerophilic workstation; Don Whitley Scientific). Lipofectamine 2000 (Invitrogen) was used for plasmid transfections.

Example 2: Cloning/Virus Production

[0051] Lentiviral pTRIPz vectors encoding Tet-inducible shRNA-GABARAPL1 and shRNA-Scrambled were purchased from Open Biosystems. For the production of viral vectors, HEK293T cells were transfected with Bug of VSV-G/envelope (addgene 8454) and pCMV(delta) R8.74/packaging (addgene 22036) and pTRIPz-GABARAPL1 or pTRIPz-SCR plasmids. Subsequently, virus containing media were collected and 2, 3 and 4 days after transfection and aliquoted. MCF7 and HT29 cells were transduced, and after selection with puromycin (4 g/mL) for 10 days, cells were pooled and analyzed. To induce knockdown, Doxycycline (1 g/mL, Sigma) was added 72 hours prior to experiments.

Example 3: Exosome Production Medium

[0052] To avoid contamination of bovine exosomes, fetal calf serum was depleted of exosomes by ultracentrifugation at 100.000g over night (16 h). Before addition to the medium, the exosome-depleted serum was filter-sterilized by a 0.22 um filter (Millipore).

Example 4: Exosome Isolation

[0053] Cells were grown until they reached 80% of confluency. To induce exosome secretion, cells were exposed to anoxia for 24 h. Exosomes were isolated from the media by differential ultracentrifugation (Beckman Coulter, sw41Ti rotor) with increasing centrifugation speeds. To remove large dead cells en cellular debris, samples were centrifuged at 300g and 16.000g for 5 and 30 minutes respectively. The pellet was thrown away. The final supernatant was centrifuged at 100.000g for 90 minutes. The exosome-containing pellet was washed with a large amount of PBS and centrifuged again at 100.000g for 90 minutes. All centrifugation steps were done at 4 C.

Example 5: Staining Endogenous GABARAPL1

[0054] Endogenous GABARAPL1 was stained by applying GABARAPL1 antibody (protein tech group, 1:50) and secondary antibody to the resuspended isolated exosomes. Exosomes were pelleted and washed with PBS. Mounted in mounting medium and visualized by fluorescence microscopy.

Example 6: Fusion Proteins/Colocalization

[0055] GABARAPL1 and LC3B PCR-fragments were subcloned (EcoRI/XhoI, New England Biolabs) into eGFP-C1 and mCherry-C1 backbone vectors (Clontech). eGFP and mCherry were fused at the N-terminal site.

Example 7: Exosome Pulldown

[0056] Isolated exosomes were incubated with GABARAPL1 antibody (1:50) for 6 hours at 4 C. Subsequently, blocked magnetic dynabeads (2 hours 1% BSN PBS-Tween, RT) were added to the exosome containing sample and incubated overnight at 4 C. at a head over head shaker. After incubation, magnetic pulldown was performed and beads were washed 3 times with PBS. Protein content was visualized by westernblot.

Example 8: Angiogenesis Array

[0057] HT29 and MCF7 GABARAPL1 knockdown and control cells were exposed to anoxia (0% 02) for 24 hours and medium was collected. Before applying conditioned medium to the angiogenesis array (Human Angiogenesis Antibody Array, Catalog #ARY007), medium was centrifuged for 10 minutes 300g. Angiogenesis array was performed according to the manufacturers manual.

Example 9: Mass Spec Sample Preparation

[0058] HT29 and MCF7 GABARAPL1 knockdown and control cells were exposed to anoxia (0.0% 02) for 24 hours. Conditioned medium (DMEM and RPMI respectively, 0% FCS) was collected and concentrated with Amicon Ultra 3K filters. Samples were visualized by Page gel electrophoresis or mass spec analysis.

Example 10: Endothelial Tube Formation Assay

[0059] Human umbilical vessel cells (HUVEC) (20.000 cells) were seeded in a matrigel coated (BD-biosciences, 50 L/well) 96-well. If applicable, cells were exposed to isolated exosomes of hypoxia (O2<0.02%) exposed tumor cells with/without anti-GABARAPL1 antibody (proteintechgroup, #10010-AP-1). Tube development was assessed after 16 hours exposure at 37 C.

Example 11: Tumor Cell Adherence

[0060] HUVEC cells were grown as monolayer in 24-well format. When indicated, HUVEC monolayers were exposed for 16 hours to isolated exosomes of hypoxia (O2<0.02%) exposed tumor cells with/without anti-GABARAPL1 antibody (proteintechgroup, #10010-AP-1). 20.000 GFP-expressing (eGFP-C1, clontech) MDA-MB-231 tumor cells were added to the monolayers. After exposure for the indicated timepoints, the monolayers were extensively washed with PBS. The number of adhering cells was quantified after trysinization by flow cytometry.

Example 12: Metastasis Development

[0061] Control cells and GABARAPL1 knockdown cells were generated using pTRIPZ vectors. One million MDA-MB-231 cells were implanted in the fat-pad of female nude mice (nu/nu NMRI, Charles River). Tumor growth was assessed using caliper measurements. After reaching a primary tumor volume of 15003, the animals were killed and the lungs examined for metastasis. The lung nodules were counted manually.

Example 13: The Role of GABARAPL1 in Exosome Function

[0062] To assess if GABARAPL1 has a direct role in hypoxia-associated exosome function, we used a capillary tube formation assay in vitro. Exposure of human umbilical cord vessel endothelial cells (HUVEC) to exosome derived from hypoxia-exposed HT29, MCF7 and U87 cells (FIG. 2A) were capable of inducing tube formation in vitro. Interestingly, compared to their respective controls, exosome derived from GABARAPL1 knockdown proved less capable of inducing tube formation (FIG. 2A). Blocking GABARAPL1 using specific antibodies efficiently blocked exosome-induced tube formation (FIG. 2B). The GABARAPL1 antibody effective against exosome-induced tube formation, was unable to prevent tube formation when growth factors (VEGF, FGF-2, IGF, EGF) were added to the culture medium directly (FIG. 2C), indicating the specificity of anti-GABARAPL1 action through inhibition of exosome function and that GABARAPL1-exosome function can be inhibited by antibody targeting.

Example 14: GABARAPL1 Exosomes Facilitate Adherence of Tumor Cells to Endothelium

[0063] As shown herein, GABARAPL1 exosomes have a profound effect on enodothelial cells. This effect can be inhibited through the use of GABARAPL1 blocking antibodies. In the progress of metastasis, adherence of metastasizing tumor cells to the vessel endothelial cells is essential. To determine whether GABARAPL1 exosomes facilitate adherence of tumor cells to endothelium, HUVEC monolayers were grown and adherence of tumor cells (fluorescently labeled MDA-MB-231) was assessed by flow cytometry. Under normal conditions, 30% of the added tumor cells adhered within 5 minutes to the endothelial cells. After 1 hour, 92% of the seeded tumor cells adhered. Surprisingly, pre-exposure of endothelial cells to isolated exosomes facilitated adherence of tumor cells (50% adhered within 5 minutes and 100% after 30 minutes), which could be inhibited by pre-exposure to exosomes in combination with GABARAPL1 blocking antibodies (20% adherence after 5 minutes and 76% after 30 minutes) (FIG. 3).

Example 15: GABARAPL1 Reduction Reduces Metastasis Development

[0064] GABARAPL1 exosomes facilitate tumor cell adhesion to endothelial cells, and important step in tumor cell extravasation and development of metastasis. To determine if tumors in the absence of GABARAPL1 show reduced capacity of metastasis development, MDA-MB-231 cells were generated with GABARAPL1 knockdown. Both control and GABARAPL1 knockdown cells were implanted orthotopically in nude mice. When the primary tumors reached 1500 mm3 in size, the animals were killed and the lungs of the animals were examined for metastasis development. On average, control tumors lead to the development of 39.3 metastasis, whereas GABARAPL1 knockdown tumors lead to development of 6.8 metastasis per animal (FIG. 4).

Example 16: The Number of GABARAPL1 Exosomes in Blood Correlate with Tumor Hypoxia

[0065] In blood of healthy volunteers, no GABARAPL1 exosomes could be detected, whereas in blood of cancer patients GABARAPL1 exosomes are frequently observed. To determine if GABARAPL1 exosomes in blood can be used as biomarker for tumor hypoxia, we analyzed plasma of non-small cell lung cancer (NSCLC) patients that were assessed for the degree of tumor hypoxia by HX4 PET-scanning (Zegers et al.). Exosomes were isolated from 1 ml of plasma. GABARAPL1 exosomes were visualized by immunochemical staining using anti-GABARAPL1 antibodies. Interestingly the number of GABARAPL1-exosomes correlated with HX4 high volume (r=0.9261, P<0.001, FIG. 5).

REFERENCES

[0066] 1. Brown, J. M. and W. R. Wilson, Exploiting tumour hypoxia in cancer treatment. Nat Rev Cancer, 2004. 4(6): p. 437-47. [0067] 2. Magagnin, M. G., M. Koritzinsky, and B. G. Wouters, Patterns of tumor oxygenation and their influence on the cellular hypoxic response and hypoxia-directed therapies. Drug Resist Updat, 2006. [0068] 3. Durand, R. E. and C. Aquino-Parsons, Non-constant tumour blood flowimplications for therapy. Acta Oncol, 2001. 40(7): p. 862-9. [0069] 4. Wouters, B. G., et al., Targeting hypoxia tolerance in cancer. Drug Resist Updat, 2004. 7(1): p. 25-40. [0070] 5. Hockel, M., et al., Association between tumor hypoxia and malignant progression in advanced cancer of the uterine cervix. Cancer Res, 1996. 56(19): p. 4509-15. [0071] 6. Nordsmark, M., et al., Prognostic value of tumor oxygenation in 397 head and neck tumors after primary radiation therapy. An international multi-center study. Radiother Oncol, 2005. 77(1): p. 18-24. [0072] 7. Brizel, D. M., et al., Tumor oxygenation predicts for the likelihood of distant metastases in human soft tissue sarcoma. Cancer Res, 1996. 56(5): p. 941-3. [0073] 8. Gort, E. H., et al., Hypoxic regulation of metastasis via hypoxia-inducible factors. Curr Mol Med, 2008. 8(1): p. 60-7. [0074] 9. Gort, E. H., et al., The TWIST1 oncogene is a direct target of hypoxia-inducible factor-2alpha. Oncogene, 2008. 27(11): p. 1501-10. [0075] 10. Theys, J., et al., E-Cadherin loss associated with EMT promotes radioresistance in human tumor cells. Radiother Oncol, 2011. 99(3): p. 392-7. [0076] 11. Yang, M. H. and K. J. Wu, TWIST activation by hypoxia inducible factor-1 (HIF-1): implications in metastasis and development. Cell Cycle, 2008. 7(14): p. 2090-6. [0077] 12. Filipazzi, P., et al., Recent advances on the role of tumor exosomes in immunosuppression and disease progression. Semin Cancer Biol, 2012. 22(4): p. 342-9. [0078] 13. Grange, C., et al., Microvesicles released from human renal cancer stem cells stimulate angiogenesis and formation of lung premetastatic niche. Cancer research, 2011. 71(15): p. 5346-56. [0079] 14. Jung, T., et al., CD44v6 dependence of premetastatic niche preparation by exosomes. Neoplasia, 2009. 11(10): p. 1093-105. [0080] 15. Peinado, H., S. Lavotshkin, and D. Lyden, The secreted factors responsible for pre-metastatic niche formation: old sayings and new thoughts. Semin Cancer Biol, 2011. 21(2): p. 139-46. [0081] 16. Salomon, C., et al., Exosomal signaling during hypoxia mediates microvascular endothelial cell migration and vasculogenesis. PloS one, 2013. 8(7): p. e68451. [0082] 17. Sceneay, J., et al., Hypoxia-driven immunosuppression contributes to the pre-metastatic niche. Oncoimmunology, 2013. 2(1): p. e22355. [0083] 18. Sceneay, J., M. J. Smyth, and A. Moller, The pre-metastatic niche: finding common ground. Cancer metastasis reviews, 2013. [0084] 19. Svensson, K. J., et al., Hypoxia triggers a proangiogenic pathway involving cancer cell microvesicles and PAR-2-mediated heparin-binding EGF signaling in endothelial cells. Proc Natl Acad Sci USA, 2011. 108(32): p. 13147-52. [0085] 20. Wong, C. C., et al., Hypoxia-inducible factor 1 is a master regulator of breast cancer metastatic niche formation. Proc Natl Acad Sci USA, 2011. 108(39): p. 16369-74. [0086] 21. Overgaard, J., Hypoxic modification of radiotherapy in squamous cell carcinoma of the head and neckA systematic review and meta-analysis. Radiother Oncol, 2011. [0087] 22. Rouschop, K. M., et al., Deregulation of cap-dependent mRNA translation increases tumour radiosensitivity through reduction of the hypoxic fraction. Radiother Oncol, 2011. 99(3): p. 385-91. [0088] 23. Rouschop, K. M., et al., PERK/eIF2alpha signaling protects therapy resistant hypoxic cells through induction of glutathione synthesis and protection against ROS. Proceedings of the National Academy of Sciences of the United States of America, 2013. 110(12): p. 4622-7. [0089] 24. Rouschop, K. M., et al., The unfolded protein response protects human tumor cells during hypoxia through regulation of the autophagy genes MAP1 LC3B and ATGS. J Clin Invest, 2010. 120(1): p. 127-41. [0090] 25. Sarkar, S., et al., Hypoxia induced impairment of NK cell cytotoxicity against multiple myeloma can be overcome by IL-2 activation of the NK cells. PloS one, 2013. 8(5): p. e64835. [0091] 26. Klionsky, D. J., et al., Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy, 2008. 4(2): p. 151-75. [0092] 27. Dubois, L. J., et al., Preclinical evaluation and validation of [18F]HX4, a promising hypoxia marker for PET imaging. Proc Natl Acad Sci USA, 2011. 108(35): p. 14620-5. [0093] 28. van Loon, J., et al., PET imaging of hypoxia using [18F]HX4: a phase I trial. Eur J Nucl Med Mol Imaging, 2010. 37(9): p. 1663-8. [0094] 29. Coyle, J. E. and D. B. Nikolov, GABARAP: lessons for synaptogenesis. Neuroscientist, 2003. 9(3): p. 205-16. [0095] 30. Chakrama, F. Z., et al., GABARAPL1 (GEC1) associates with autophagic vesicles.

[0096] Autophagy, 2010. 6(4). [0097] 31. Kabeya, Y., et al., LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation. J Cell Sci, 2004. 117(Pt 13): p. 2805-12. [0098] 32. Schwarten, M., et al., Nix directly binds to GABARAP: a possible crosstalk between apoptosis and autophagy. Autophagy, 2009. 5(5): p. 690-8. [0099] 33. Novak, I., et al., Nix is a selective autophagy receptor for mitochondrial clearance. EMBO Rep. 11(1): p. 45-51. [0100] 34. Betin, V. M. and J. D. Lane, Caspase cleavage of Atg4D stimulates GABARAP-L1 processing and triggers mitochondrial targeting and apoptosis. J Cell Sci, 2009. 122(Pt 14): p. 2554-66. [0101] 35. Ballot, G., et al., Hypoxia-induced autophagy is mediated through hypoxia-inducible factor induction of BNIP3 and BNIP3L via their BH3 domains. Mol Cell Biol, 2009. 29(10): p. 2570-81. [0102] 36. Zhang, J. and P. A. Ney, Role of BNIP3 and NIX in cell death, autophagy, and mitophagy. Cell Death Differ, 2009. 16(7): p. 939-46. [0103] 37. Biaoxue, R., et al., Upregulation of Hsp90-beta and annexin A1 correlates with poor survival and lymphatic metastasis in lung cancer patients. Journal of experimental & clinical cancer research: CR, 2012. 31: p. 70. [0104] 38. Wang, J., et al., High expression of heat shock protein 90 is associated with tumor aggressiveness and poor prognosis in patients with advanced gastric cancer. PloS one, 2013. 8(4): p. e62876. [0105] 39. Mazzocca, A., F. Liotta, and V. Carloni, Tetraspanin CD81-regulated cell motility plays a critical role in intrahepatic metastasis of hepatocellular carcinoma. Gastroenterology, 2008. 135(1): p. 244-256 el. [0106] 40. Trojan, L., et al., Identification of metastasis-associated genes in prostate cancer by genetic profiling of human prostate cancer cell lines. Anticancer Res, 2005. 25(1A): p. 183-91. [0107] 41. Kita, Y., et al., STC2: a predictive marker for lymph node metastasis in esophageal squamous-cell carcinoma. Annals of surgical oncology, 2011. 18(1): p. 261-72. [0108] 42. Pena, C., et al., STC1 expression by cancer-associated fibroblasts drives metastasis of colorectal cancer. Cancer research, 2013. 73(4): p. 1287-97. [0109] 43. Volland, S., et al., Stanniocalcin 2 promotes invasion and is associated with metastatic stages in neuroblastoma. International journal of cancer. Journal international du cancer, 2009. 125(9): p. 2049-57. [0110] 44. Katchman, B. A., et al., Expression of quiescin sulfhydryl oxidase 1 is associated with a highly invasive phenotype and correlates with a poor prognosis in Luminal B breast cancer. Breast cancer research: BCR, 2013. 15(2): p. R28. [0111] 45. Meding, S., et al., Tissue-based proteomics reveals FXYD3, S100A11 and GSTM3 as novel markers for regional lymph node metastasis in colon cancer. The Journal of pathology, 2012. [0112] 46. Nipp, M., et al., S100-A10, thioredoxin, and S100-A6 as biomarkers of papillary thyroid carcinoma with lymph node metastasis identified by MALDI imaging. Journal of molecular medicine, 2012. 90(2): p. 163-74. [0113] 47. Hibino, S., et al., Identification of an active site on the laminin alpha5 chain globular domain that binds to CD44 and inhibits malignancy. Cancer research, 2004. 64(14): p. 4810-6. [0114] 48. Chen, J., et al., Proteome analysis of gastric cancer metastasis by two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-mass spectrometry for identification of metastasis-related proteins. Journal of proteome research, 2004. 3(5): p. 1009-16. [0115] 49. Gibert, B., et al., Targeting heat shock protein 27 (HspB1) interferes with bone metastasis and tumour formation in vivo. Br J Cancer, 2012. 107(1): p. 63-70. [0116] 50. Guo, K., et al., Regulation of HSP27 on NF-kappaB pathway activation may be involved in metastatic hepatocellular carcinoma cells apoptosis. BMC Cancer, 2009. 9: p. 100. [0117] 51. Nagaraja, G. M., P. Kaur, and A. Asea, Role of human and mouse HspB1 in metastasis. Curr Mol Med, 2012. 12(9): p. 1142-50. [0118] 52. Shiota, M., et al., Hsp27 regulates epithelial mesenchymal transition, metastasis, and circulating tumor cells in prostate cancer. Cancer Res, 2013. 73(10): p. 3109-19. [0119] 53. Song, H. Y., et al., [Proteomic analysis on metastasis-associated proteins of hepatocellular carcinoma tissues]. Zhonghua Gan Zang Bing Za Zhi, 2005. 13(5): p. 331-4. [0120] 54. Song, H. Y., et al., Proteomic analysis on metastasis-associated proteins of human hepatocellular carcinoma tissues. J Cancer Res Clin Oncol, 2006. 132(2): p. 92-8. [0121] 55. Wei, L., et al., Hsp27 participates in the maintenance of breast cancer stem cells through regulation of epithelial-mesenchymal transition and nuclear factor-kappaB. Breast Cancer Res, 2011. 13(5): p. R101. [0122] 56. Zhang, D., L. L. Wong, and E. S. Koay, Phosphorylation of Ser78 of Hsp27 correlated with HER-2/neu status and lymph node positivity in breast cancer. Molecular cancer, 2007. 6: p. 52. [0123] 57. Zhao, L., et al., Differential proteomic analysis of human colorectal carcinoma cell lines metastasis-associated proteins. J Cancer Res Clin Oncol, 2007. 133(10): p. 771-82. [0124] 58. Zhao, M., et al., Increased expression of heat shock protein 27 correlates with peritoneal metastasis in epithelial ovarian cancer. Reprod Sci, 2012. 19(7): p. 748-53. [0125] 59. Coutinho-Camillo, C. M., et al., Nucleophosmin, p53, and Ki-67 expression patterns on an oral squamous cell carcinoma tissue microarray. Hum Pathol, 2010. 41(8): p. 1079-86. [0126] 60. Liu, Y., et al., Expression of nucleophosmin/NPM1 correlates with migration and invasiveness of colon cancer cells. J Biomed Sci, 2012. 19: p. 53. [0127] 61. Walsh, N., et al., RNAi knockdown of Hop (Hsp70/Hsp90 organising protein) decreases invasion via MMP-2 down regulation. Cancer Lett, 2011. 306(2): p. 180-9. [0128] 62. Walsh, N., et al., Identification of pancreatic cancer invasion-related proteins by proteomic analysis. Proteome Sci, 2009. 7: p. 3. [0129] 63. van den Boom, J. G., et al., Exosomes as nucleic acid nanocarriers. Advanced drug delivery reviews, 2013. 65(3): p. 331-5. [0130] 64. Holzel, M., A. Bovier, and T. Tuting, Plasticity of tumour and immune cells: a source of heterogeneity and a cause for therapy resistance? Nature reviews. Cancer, 2013. 13(5): p. 365-76. [0131] 65. Park, J. E., et al., Hypoxic tumor cell modulates its microenvironment to enhance angiogenic and metastatic potential by secretion of proteins and exosomes. Molecular & cellular proteomics: MCP, 2010. 9(6): p. 1085-99. [0132] 66. Azmi, A. S., B. Bao, and F. H. Sarkar, Exosomes in cancer development, metastasis, and drug resistance: a comprehensive review. Cancer metastasis reviews, 2013. [0133] 67. King, H. W., M. Z. Michael, and J. M. Gleadle, Hypoxic enhancement of exosome release by breast cancer cells. BMC Cancer, 2012. 12: p. 421. [0134] 68. Kucharzewska, P., et al., Exosomes reflect the hypoxic status of glioma cells and mediate hypoxia-dependent activation of vascular cells during tumor development. Proceedings of the National Academy of Sciences of the United States of America, 2013. 110(18): p. 7312-7. [0135] 69. Karreman, M. A., et al., Optimizing immuno-labeling for correlative fluorescence and electron microscopy on a single specimen. Journal of structural biology, 2012. 180(2): p. 382-6. [0136] 70. Schaaf, M. B., et al., The autophagy associated gene, ULK1, promotes tolerance to chronic and acute hypoxia. Radiotherapy and oncology: journal of the European Society for Therapeutic Radiology and Oncology, 2013. [0137] 71. Jutten, B., et al., EGFR overexpressing cells and tumors are dependent on autophagy for growth and survival. Radiotherapy and oncology: journal of the European Society for Therapeutic Radiology and Oncology, 2013. [0138] 72. Liu, Q., et al., TH-302, a hypoxia-activated prodrug with broad in vivo preclinical combination therapy efficacy: optimization of dosing regimens and schedules. Cancer Chemother Pharmacol, 2012. 69(6): p. 1487-98. [0139] 73. Meng, F., et al., Molecular and cellular pharmacology of the hypoxia-activated prodrug TH-302. Mol Cancer Ther, 2012. 11(3): p. 740-51. [0140] 74. Sun, J. D., et al., Selective tumor hypoxia targeting by hypoxia-activated prodrug TH-302 inhibits tumor growth in preclinical models of cancer. Clin Cancer Res, 2012. 18(3): p. 758-70. [0141] 75. Jemal, A., et al., Global cancer statistics. CA: a cancer journal for clinicians, 2011. 61(2): p. 69-90. [0142] 76. Bacia, K., S. A. Kim, and P. Schwille, Fluorescence cross-correlation spectroscopy in living cells. Nat Methods, 2006. 3(2): p. 83-9. [0143] 77. Vaupel, P., et al., Oxygenation of human tumors: evaluation of tissue oxygen distribution in breast cancers by computerized 02 tension measurements. Cancer Res, 1991. 51(12): p. 3316-22. [0144] 78. Fyles, A., et al., Tumor hypoxia has independent predictor impact only in patients with node-negative cervix cancer. Journal of clinical oncology: official journal of the American Society of Clinical Oncology, 2002. 20(3): p. 680-7. [0145] 79. Pitson, G., et al., Tumor size and oxygenation are independent predictors of nodal diseases in patients with cervix cancer. International journal of radiation oncology, biology, physics, 2001. 51(3): p. 699-703. [0146] 80. Sundfor, K., H. Lyng, and E. K. Rofstad, Tumour hypoxia and vascular density as predictors of metastasis in squamous cell carcinoma of the uterine cervix. British journal of cancer, 1998. 78(6): p. 822-7. [0147] 81. Milosevic, M., et al., Tumor hypoxia predicts biochemical failure following radiotherapy for clinically localized prostate cancer. Clinical cancer research: an official journal of the American Association for Cancer Research, 2012. 18(7): p. 2108-14. [0148] 82. Rouschop, K. M., et al., Autophagy is required during cycling hypoxia to lower production of reactive oxygen species. Radiother Oncol, 2009. 92(3): p. 411-6. [0149] 83. Klionsky, D. J., et al., Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy, 2012. 8(4): p. 445-544. [0150] 84. Klionsky, D. J. et al., Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy. 2008 February; 4(2):151-75. [0151] 85. Keulers et al., Abstractbook 13th international Wolfsberg meeting on molecular Radiation Biology/oncology, Ermatingen Switzerland, Jun. 22-24, 2013, p 88. [0152] 86. Sceneay, J. et al., The pre-metastatic niche: finding common ground Cancer Metastasis Rev. 2013 December; 32(3-4):449-64. [0153] 87. Zegers C M, van Elmpt W, Reymen B, Even A J, Troost E G, Oilers M C, Hoebers F J, Houben R M, Eriksson J, Windhorst A D, Mottaghy F M, De Ruysscher D, Lambin P. In Vivo Quantification of Hypoxic and Metabolic Status of NSCLC Tumors Using [18F]HX4 and [18F]FDG-PET/CT Imaging. Clin Cancer Res. 2014 Dec. 15; 20(24):6389-97. [0154] 88. Valadi, H. et al., Nature Cell Biol. (2007) 9: 654-659 and supplementary information, table S2.