Application of Radiotherapy and Chemotherapy Protocols to Pre‐Clinical Tumor Models

Randy Burd1, Phyllis Wachsberger2

1 University of Arizona, Tucson, Arizona, 2 Thomas Jefferson University, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 14.7
DOI:  10.1002/0471141755.ph1407s38
Online Posting Date:  September, 2007
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit (1) provides background into understanding how agents that target specific molecules or receptors (molecular‐targeted agents), in particular, agents affecting the tumor vasculature (perivasculature network in tumors), interact with and modify radiation therapy; (2) details factors affecting interpretation of results in murine tumor model experiments utilizing radiation therapy and drug combinations; and (3) provides specific protocols for the application of radiation therapy, both alone and in combination with chemotherapy and/or molecular‐targeted agents. Curr. Protoc. Pharmacol. 38:14.7.1‐14.7.24. © 2007 by John Wiley & Sons, Inc.

Keywords: chemotherapy; radiation therapy; tumor models; tumor hypoxia; tumor growth delay; preclinical

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Tumors for Hind Limb Growth
  • Basic Protocol 2: Preparation of Cells for Tumor Inoculation Using Cellstripper
  • Basic Protocol 3: Tumor Cell Inoculation and Radiation Therapy
  • Alternate Protocol 1: Determination of 50% Tumor Control Dose (TCD50)
  • Basic Protocol 4: Treatment Sequencing of an Antiangiogenic Plus Radiation Therapy to Determine Optimal Tumor Growth Delay
  • Basic Protocol 5: Trimodal Treatment with the Angiogenesis Inhibitor Vandetanib, Radiation Therapy, and the Chemotherapeutic CPT‐11
  • Basic Protocol 6: Measurement of Tumor Hypoxia Using the Oxylite Fiber Optic Probe
  • Basic Protocol 7: Measurement of Tumor Hypoxia Using the Hypoxia Marker, EF5
  • Basic Protocol 8: FDG PET Imaging to Determine Metabolically Inactive Fraction of Tumor
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Preparation of Tumors for Hind Limb Growth

  Materials
  • Tumor cell suspension (see protocol 2)
  • Mice of appropriate strain (4 to 8 weeks old; see Strategic Planning)
  • 1× PBS containing 0.1% gentamicin solution, ice cold
  • 70% ethanol
  • Medium containing 10% FBS/0.1% gentamicin solution, ice cold
  • Digital calipers (Fisher Scientific or VWR)
  • Sterile pads
  • Sterile surgical instruments (forceps, scissors)
  • Petri dishes
  • Disposable scalpels (curved end)
  • 2 × 2 × 2–mm metal measuring grid (Fisher Scientific)
  • 25‐G trocar
  • 50‐ml centrifuge tubes
  • Cell strainer or plastic mesh to cover tubes (Fisher Scientific)
  • 25‐ml pipets
  • Rubber cell scraper
  • 17‐G blunt‐end needle
  • 175‐cm2 flasks
  • 37°C, 5% CO 2 humidified incubator

Basic Protocol 2: Preparation of Cells for Tumor Inoculation Using Cellstripper

  Materials
  • Serum‐free growth medium
  • 1× PBS without calcium and magnesium
  • Tumor cells grown as a monolayer (80% confluent) in a 175‐cm2 flask (see protocol 1)
  • Cellstripper solution (Mediatech), 37°C
  • 0.4% trypan blue
  • 37°C incubator
  • 50‐ml centrifuge tubes
  • Hemacytometer
  • 17‐G blunt‐end needle

Basic Protocol 3: Tumor Cell Inoculation and Radiation Therapy

  Materials
  • Mice of appropriate strain (see Strategic Planning)
  • Pentobarbital or isoflurane and vaporizer (Viking Medical)
  • 1 × 107 cells/ml single‐cell suspension of tumor cells (see protocol 2)
  • Ketamine
  • Xylazine
  • BL‐2 level laminar flow hood
  • Nose cone
  • 1‐ml syringe
  • 25‐G needle
  • Heating pad and pump (Viking Medical)
  • Digital calipers (Fisher Scientific or VWR)
  • Lead shield (Figs. and ; lead sheeting available from Radiation Protection Products)
  • Radiation source (Precision X‐Ray; Rad Source Technologies; MDS Nordion)

Alternate Protocol 1: Determination of 50% Tumor Control Dose (TCD50)

  Materials
  • U87 human glioblastoma cells (ATCC # HTB‐14)
  • Homozygous nCRNU mice (males, 5 to 8 weeks old; Taconic Farms)
  • Ketamine
  • Xylazine
  • Experimental antiangiogenic compound
  • Leadshield (Figs. and ; lead sheeting available from Radiation Protection Products)
  • Radiation source (Precision X‐Ray; Rad Source Technologies; MDS Nordion)

Basic Protocol 4: Treatment Sequencing of an Antiangiogenic Plus Radiation Therapy to Determine Optimal Tumor Growth Delay

  Materials
  • nCRNU mice (Taconic Farms)
  • LoVo colorectal adenocarcinoma cells (ATCC #CCL‐229)
  • Ketamine
  • Xylazine
  • CPT‐11 (Pfizer Pharmaceuticals) prepared in deionized water containing 0.018% lactic acid and 9.0% sorbitol
  • Experimental antiangiogenic compound (e.g., vandetanib; Astra Zeneca)
  • Radiation source (Precision X‐Ray; Rad Source Technologies; MDS Nordion)
  • Lead shield (Figs. and ; lead sheeting available from Radiation Protection Products)

Basic Protocol 5: Trimodal Treatment with the Angiogenesis Inhibitor Vandetanib, Radiation Therapy, and the Chemotherapeutic CPT‐11

  Materials
  • Tumored mice
  • Ketamine, xylazine, or isoflurane and vaporizer (Viking Medical)
  • Acepromazine (Butler Animal Health Supply)
  • Heating pad and pump (Viking Medical)
  • Thermometer
  • 27.5‐G needles
  • Oxygen probes with temperature sensor (30‐G; Oxford Optronix)
  • OxyLite (Oxford Optronix)
  • Micromanipulator (e.g., Märzhäuser Wetzlar GmbH)

Basic Protocol 6: Measurement of Tumor Hypoxia Using the Oxylite Fiber Optic Probe

  Materials
  • 20 mg/ml Hypoxyprobe‐1 (Chemicon) in normal (0.9%) saline
  • Tumored animals (treated)
  • Tissue Tek OTC compound (EMS)
  • 2‐methylbutane (Sigma‐Aldrich)
  • Dry ice
  • 4% paraformaldehyde (Sigma‐Aldrich)
  • 1× PBS, ice cold
  • Tween 20 (Sigma‐Aldrich)
  • Bovine serum albumin (BSA)
  • Fat‐free milk
  • Normal mouse serum
  • Hypoxyprobe‐1 MAb1 (Chemicon)
  • Fluorescent 20 Antibody (anti‐mouse; Abcam)
  • Freezing cassette
  • Cryostat (Jencons Scientific)

Basic Protocol 7: Measurement of Tumor Hypoxia Using the Hypoxia Marker, EF5

  Materials
  • Mouse subject
  • Ketamine
  • Acepromazine (Butler Animal Health Supply)
  • 18‐Fluorodeoxyglucose (18F‐FDG; IBA Molecular)
  • 50‐ml specimen tubes
  • Access to PET imager
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Ansiaux, R., Baudelet, C., Jordan, B.F., Crokart, N., Martinive, P., DeWever, J., Gregoire, V., Feron, O., and Gallez, B. 2006. Mechanism of reoxygenation after antiangiogenic therapy using SU5416 and its importance for guiding combined antitumor therapy. Cancer Res. 66:9698‐9704.
   Belani, C.P., Choy, H., Bonomi, P., Scott, C., Travis, P., Haluschak, J., and Curran, W.J. Jr., 2005. Combined chemoradiotherapy regimens of paclitaxel and carboplatin for locally advanced non‐small‐cell lung cancer: A randomized phase II locally advanced multi‐modality protocol. J. Clin. Oncol. 23:5883‐5891.
   Benjamin, L.E., Golijanin, D., Itin, A., Pode, D., and Keshet, E. 1999. Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal. J. Clin. Invest 103:159‐165.
   Biedermann, K.A., Sun, J.R., Giaccia, A.J., Tosto, L.M., and Brown, J.M. 1991. SCID mutation in mice confers hypersensitivity to ionizing radiation and a deficiency in DNA double‐strand break repair. Proc. Natl. Acad. Sci. U.S.A 88:1394‐1397.
   Bosma, M.J. and Carroll, A.M. 1991. The SCID mouse mutant: Definition, characterization, and potential uses. Annu. Rev. Immunol. 9:323‐350.
   Brizel, D.M., Scully, S.P., Harrelson, J.M., Layfield, L.J., Bean, J.M., Prosnitz, L.R., and Dewhirst, M.W. 1996. Tumor oxygenation predicts for the likelihood of distant metastases in human soft tissue sarcoma. Cancer Res. 56:941‐943.
   Brizel, D.M., Sibley, G.S., Prosnitz, L.R., Scher, R.L., and Dewhirst, M.W. 1997. Tumor hypoxia adversely affects the prognosis of carcinoma of the head and neck. Int. J. Radiat. Oncol. Biol. Phys. 38:285‐289.
   Burd, R., Lavorgna, S.N., Daskalakis, C., Wachsberger, P.R., Wahl, M.L., Biaglow, J.E., Stevens, C.W., and Leeper, D.B. 2003. Tumor oxygenation and acidification are increased in melanoma xenografts after exposure to hyperglycemia and meta‐iodo‐benzylguanidine. Radiat. Res. 159:328‐335.
   Evans, S.M., Hahn, S., Pook, D.R., Jenkins, W.T., Chalian, A.A., Zhang, P., Stevens, C., Weber, R., Weinstein, G., Benjamin, I., Mirza, N., Morgan, M., Rubin, S., McKenna, W.G., Lord, E.M., and Koch, C.J. 2000. Detection of hypoxia in human squamous cell carcinoma by EF5 binding. Cancer Res. 60:2018‐2024.
   Folkman, J., Merler, E., Abernathy, C., and Williams, G. 1971. Isolation of a tumor factor responsible for angiogenesis. J. Exp. Med. 133:275‐288.
   Fujita, Y., Habazettl, H., Corso, C.O., Messmer, K., and Yada, T. 1997. Comparative effects of hypotension due to isoflurane, nitroglycerin, and adenosine on subendocardial microcirculation: Observation of the in situ beating swine heart under critical stenosis. Anesthesiology 87:343‐353.
   Furuta, Y., Hunter, N., Barkley, T.J., Hall, E., and Milas, L. 1988. Increase in radioresponse of murine tumors by treatment with indomethacin. Cancer Res. 48:3008‐3013.
   Gorski, D.H., Beckett, M.A., Jaskowiak, N.T., Calvin, D.P., Mauceri, H.J., and Salloum, R.M. 1999. Blockage of the vascular endothelial growth factor stress response increases the antitumor effects of ionizing radiation. Cancer Res. 59:3374‐3378.
   Hall, E.J. and Giaccia, A.J. 2006. Oxygen effect. In Radiobiology for the Radiologist, 6th ed. (L. McAllister, L. Bierig, and K. Barrett, eds.), pp. 85‐105. Lippincott, Williams & Wilkins, Philadelphia.
   Holash, J., Davis, S., Papadopoulos, N., Croll, S.D., Ho, L., Russell, M., Boland, P., Leidich, R., Hylton, D., Burova, E., Ioffe, E., Huang, T., Radziejewski, C., Bailey, K., Fandl, J.P., Daly, T., Wiegand, S.J., Yancopoulos, G.D., and Rudge, J.S. 2002. VEGF‐Trap: A VEGF blocker with potent antitumor effects. Proc. Natl. Acad. Sci. U.S.A 99:11393‐11398.
   Huber, P.E., Bischof, M., Jenne, J., Heiland, S., Peschke, P., Saffrich, R., Grone, H.J., Debus, J., Lipson, K.E., and Abdollahi, A. 2005. Trimodal cancer treatment: Beneficial effects of combined antiangiogenesis, radiation, and chemotherapy. Cancer Res. 65:3643‐3655.
   Inai, T., Mancuso, M., Hashizume, H., Baffert, F., Haskell, A., Baluk, P., Hu‐Lowe, D.D., Shalinsky, D.R., Thurston, G., Yancopoulos, G.D., and McDonald, D.M. 2004. Inhibition of vascular endothelial growth factor (VEGF) signaling in cancer causes loss of endothelial fenestrations, regression of tumor vessels, and appearance of basement membrane ghosts. Am. J. Pathol. 165:35‐52.
   Jain, R.K. 2005. Normalization of tumor vasculature: An emerging concept in antiangiogenic therapy. Science 307:58‐62.
   Kapinya, K.J., Prass, K., and Dirnagl, U. 2002. Isoflurane induced prolonged protection against cerebral ischemia in mice: A redox sensitive mechanism? Neuroreport 13:1431‐1435.
   Kishi, K., Petersen, S., Petersen, C., Hunter, N., Mason, K., Masferrer, J.L., Tofilon, P.J., and Milas, L. 2000. Preferential enhancement of tumor radioresponse by a cyclooxygenase‐2 inhibitor. Cancer Res. 60:1326‐1331.
   Koch, C.J., Evans, S.M., and Lord, E.M. 1995. Oxygen dependence of cellular uptake of EF5 [2‐(2‐nitro‐1H‐imidazol‐1‐yl)‐N‐(2,2,3,3,3‐pentafluoropropyl)a cet amide]: Analysis of drug adducts by fluorescent antibodies vs bound radioactivity. Br. J. Cancer 72:869‐874.
   Lee, C.G., Heijn, M., di, T.E., Griffon‐Etienne, G., Ancukiewicz, M., Koike, C., Park, K.R., Ferrara, N., Jain, R.K., Suit, H.D., and Boucher, Y. 2000. Anti‐vascular endothelial growth factor treatment augments tumor radiation response under normoxic or hypoxic conditions. Cancer Res. 60:5565‐5570.
   Lei, H., Grinberg, O., Nwaigwe, C.I., Hou, H.G., Williams, H., Swartz, H.M., and Dunn, J.F., 2001. The effects of ketamine‐xylazine anesthesia on cerebral blood flow and oxygenation observed using nuclear magnetic resonance perfusion imaging and electron paramagnetic resonance oximetry. Brain Res. 913:174‐179.
   Milas, L., Fang, F.M., Mason, K.A., Valdecanas, D., Hunter, N., Koto, M., and Ang, K.K. 2007. Importance of maintenance therapy in C225‐induced enhancement of tumor control by fractionated radiation. Int. J. Radiat. Oncol. Biol. Phys. 67:568‐572.
   Moeller, B.J., Cao, Y., Li, C.Y., and Dewhirst, M.W. 2004. Radiation activates HIF‐1 to regulate vascular radiosensitivity in tumors: Role of reoxygenation, free radicals, and stress granules. Cancer Cell 5:429‐441.
   Moulder, J.E. and Rockwell, S. 1984. Hypoxic fractions of solid tumors: Experimental techniques, methods of analysis, and a survey of existing data. Int. J. Radiat. Oncol. Biol. Phys. 10:695‐712.
   Nehls, M., Pfeifer, D., Schorpp, M., Hedrich, H., and Boehm, T. 1994. New member of the winged‐helix protein family disrupted in mouse and rat nude mutations. Nature 372:103‐107.
   Nias, A.H. and Perry, P.M. 1989. Variation of tumour radiosensitivity with time after anaesthetic. Br. J. Radiol. 62:932‐935.
   Nieder, C., Wiedenmann, N., Andratschke, N., and Molls, M. 2006. Current status of angiogenesis inhibitors combined with radiation therapy. Cancer Treat. Rev. 32:348‐364.
   Ryan, A.J. and Wedge, S.R. 2005. ZD6474‐a novel inhibitor of VEGFR and EGFR tyrosine kinase activity. Br. J. Cancer 92:S6‐S13.
   Sakakibara, T., Xu, Y., Bumpers, H.L., Chen, F.A., Bankert, R.B., Arredondo, M.A., Edge, S.B., and Repasky, E.A. 1996. Growth and metastasis of surgical specimens of human breast carcinomas in SCID mice. Cancer J. Sci. Am. 2:291‐300.
   Solomon, B., Binns, D., Roselt, P., Weibe, L.I., McArthur, G.A., Cullinane, C., and Hicks, R.J. 2005. Modulation of intratumoral hypoxia by the epidermal growth factor receptor inhibitor gefitinib detected using small animal PET imaging. Mol. Cancer Ther. 4:1417‐1422.
   Suit, H.D., Sedlacek, R.S., Silver, G., and Dosoretz, D. 1985. Pentobarbital anesthesia and the response of tumor and normal tissue in the C3Hf/sed mouse to radiation. Radiat. Res. 104:47‐65.
   Taghian, A., Budach, W., Zietman, A., Freeman, J., Gioioso, D., Ruka, W., and Suit, H.D. 1993. Quantitative comparison between the transplantability of human and murine tumors into the subcutaneous tissue of NCr/Sed‐nu/nu nude and severe combined immunodeficient mice. Cancer Res. 53:5012‐5017.
   Tong, R.T., Boucher, Y., Kozin, S.V., Winkler, F., Hicklin, D.J., and Jain, R.K. 2004. Vascular normalization by vascular endothelial growth factor receptor 2 blockade induces a pressure gradient across the vasculature and improves drug penetration in tumors. Cancer Res. 64:3731‐3736.
   Urano, M., Chen, Y., Humm, J., Koutcher, J.A., Zanzonico, P., and Ling, C. 2002. Measurements of tumor tissue oxygen tension using a time‐resolved luminescence‐based optical oxylite probe: Comparison with a paired survival assay. Radiat. Res. 158:167‐173.
   Vanhoefer, U., Harstrick, A., Achterrath, W., Cao, S., Seeber, S., and Rustum, Y.M. 2001. Irinotecan in the treatment of colorectal cancer: Clinical overview. J. Clin. Oncol 19:1501‐1518.
   Wachsberger, P., Burd, R., and Dicker, A.P. 2003. Tumor response to ionizing radiation combined with antiangiogenesis or vascular targeting agents: Exploring mechanisms of interaction. Clin. Cancer Res. 9:1957‐1971.
   Wachsberger, P., Burd, R., and Dicker, A.P. 2004. Improving tumor response to radiotherapy by targeting angiogenesis signaling pathways. Hematol. Oncol. Clin. North Am. 18:1039‐1057.
   Wachsberger, P.R., Burd, R., Marero, N., Daskalakis, C., Ryan, A., McCue, P., and Dicker, A.P. 2005. Effect of the tumor vascular‐damaging agent, ZD6126, on the radioresponse of U87 glioblastoma. Clin. Cancer Res. 11:835‐842.
   Wachsberger, P.R., Burd, R., Strickler, T., Kulp, K., Ryan, A.J., and Dicker, A.P. 2006. Improved antitumor activity by combining vandetanib (Zactima; ZD6474) with radiotherapy and irinotecan in the LoVo human colorectan cancer xenograpt model. In EORTC/NCI‐AACR Annual Meeting.
   Wachsberger, P.R., Burd, R., Cardi, C., Thakur, M., Daskalakis, C., Holash, J., Yancopoulos, G.D., and Dicker, A.P. 2007. VEGF Trap in combination with radiotherapy improves tumor control in U87 glioblastoma. Int. J. Radiat. Oncol. Biol. Phys. 67:1526‐1537.
   Wen, B., Urano, M., O'Donoghue, J.A., and Ling, C.C. 2006. Measurements of partial oxygen pressure pO2 using the OxyLite system in R3327‐AT tumors under isoflurane anesthesia. Radiat. Res. 166:512‐518.
   Winkler, F., Kozin, S.V., Tong, R.T., Chae, S.S., Booth, M.F., Garkavtsev, I., Xu, L., Hicklin, D.J., Fukumura, D., di Tomaso, E., Munn, L.L., and Jain, R.K. 2004. Kinetics of vascular normalization by VEGFR2 blockade governs brain tumor response to radiation: Role of oxygenation, angiopoietin‐1, and matrix metalloproteinases. Cancer Cell 6:553‐563.
   Yaromina, A., Holscher, T., Eicheler, W., Rosner, A., Krause, M., Hessel, F., Petersen, C., Thames, H.D., Baumann, M., and Zips, D. 2005. Does heterogeneity of pimonidazole labelling correspond to the heterogeneity of radiation‐response of FaDu human squamous cell carcinoma? Radiother.Oncol. 76:206‐212.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library