Studies of Radiochemotherapy in an Orthotopic Cervix Cancer Model. R.P. Hill, N. Chaudary, D.W. Hedley, S, Jelveh, P. Lindsay, H. Mackay, M. Milosevic. Ontario Cancer Institute and Princess Margaret Cancer Centre, University Health Network, and

University of Toronto, Toronto, Ontario, Canada.

Methods Cervix Orthotopic Xenograft Model Small pieces of ME180 human cervix cancer xenografts were used for implantation into the cervix of NOD/SCID, NOD/Rag1-/- /gammaT null (NRG) mice or RAG2/gammaT null mice. Mice were monitored weekly by palpation for tumor growth. Tumors are imaged at ~4-5mm in diameter (see below) and assigned to the treatment groups. At sacrifice para-aortic lymph node metastases were assessed using the DsRed fluorescence marker in the ME180 cells then excised and metastasis confirmed with H&E staining. Irradiated intestinal tissue was excised for histological analysis. In vivo cyclic hypoxia Mice bearing orthotopic tumors were exposed to cyclic hypoxia (12 x 10min cycles of breathing 7%O2 or air; 4hr/day/2 wks) during their growth with or without receiving 5E1 for assessment of effects on lymph node metastases. 5E1 Treatment 5E1, an antibody that blocks the SHH and IHH ligands was administered by i.p injection (20 mg/kg/weekly) with cyclic hypoxia or fractionated irradiation. (see below). Irradiation and Cisplatin Treatment. Fractionated radiation treatment was given to the orthotopic xenografts using an image-guided small animal irradiation. (see Figure 1) using an 8-beam configuration with an 8mm collimator. Each animal was set-up individually for each treatment and imaged and adjusted to located the tumour in the beam. Fractionated treatment was delivered 3x/wk (5Gy fractions) or 5x/wk (2 Gy fractions) with or without Cisplatin given weekly (8mg/kg) or 5 days/wk (2mg/kg/day). Tumor size was monitored 2x/wk using the imaging facility of the irradiator.

Conclusions 1) The combination of a small animal image-guided irradiator with an orthotopic xenograft model allows for preclinical studies with radiochemotherapy that can closely mimic clinical treatments. 2) The combination treatment with fractionated irradiation and the hedgehog pathway inhibitor (5E1) shows increased efficacy consistent with an additive effect of the two agents. This agent also significantly affects the development of lymphnode metastasis in the mice, further supporting the potential value of Hedgehog inhibitors for combination with radiation in the treatment of cervix cancer. 3) The treatments involving fractionated irradiation and cisplatin show limited evidence for additive effects but the data is limited by toxicity associated with multiple doses of cisplatin combined with radiation. Further studies are required with lower doses more equivalent to current therapy doses (~ 6mg/kg/wk).

Abstract: We have used a small animal irradiator/imager to deliver fractionated dose treatment and chemotherapy to orthotopically growing human cervix cancer xenografts in immune-deprived mice. Methods: Irradiation treatment has been delivered to tumours of 5-8 mm diameter growing in the cervix of mice using an 8-beam protocol with imaging of the target immediately prior to each fraction. The treatment plan allows for a 1-2 mm margin around the imaged tumour target. The radiation treatment (225kVp X-rays) is being combined with cisplatin to match current external beam treatment procedures for cervix cancers. Tumour growth delay analysis is being performed using the imaging features of the irradiator to assess tumour size as a function of time during and after the treatment. We are testing additional combination treatment with a Hedgehog pathway inhibitor, since our recent studies show that this pathway is significantly upregulated in many cervix cancer cancers. Results: There is limited response to single dose treatment with cisplatin (12 mg/kg) alone but no enhanced response when combined with fractionated irradiation. Ongoing experiments are testing daily 2Gy fractions combined with 5E1, an antibody that blocks SHH and IHH activity. Growth of nodal metastases in the aortic chain is also reduced by this Hh inhibitor. Normal tissues are being collected at the time of sacrifice of the mice for histological analyses (data not yet available). Conclusions: This combination of a small animal irradiator/imager with an orthotopic xenograft model allows for preclinical studies with radiochemotherapy that can closely mimic clinical studies.

Figure 1: (A)Treatment with 5E1 antibody reduces expression of Hh pathway genes in ME180 tumors. Treatment with 5E1 during tumor growth significantly reduces the expression of both human (tumor cells) and mouse (stroma) Hedgehog pathway genes as assessed by RT-PCR of mRNA extracted from whole tumor tissue. Mouse genes are low presumably because of low levels of stroma in the ME180 tumors. B) Treatment with 5E1 reduces tumor growth and lymph node metastasis under normoxia (air) or cyclic hypoxia. As observed previously exposure to cyclic hypoxia causes reduced tumor growth but increased metastasis in the orthotopic model. The tumor growth is further reduced by blocking Hh signalling by 5E1 treatment and the increase in metastasis is also severely depressed.

Figure 2: Results from fractionated radiation treatment of ME180 orthotopic xenografts in combination with cisplatin or 5E1 treatment. A) Growth curves for tumours treated with 3x5Gy given over 1 wk with a single dose of cisplatin (12 mg/kg) given 1 hr before the first fraction. Both treatments affect tumour growth but there is no evidence for increased efficacy with the combination treatment. B) Growth curves for tumours treated with 2Gy fractions given 5 days/wk over 2 wks (total 20 Gy) with cisplatin (8 mg/kg given weekly before the 1st and 6th fraction) or 2mg/kg given daily 1 hr before each fraction. The results show little evidence for increased efficacy of the combination. C) Growth curves for tumours treated with 2Gy fractions given over 3 wks (total 30 Gy) with 5E1 (20 mg/kg given weekly for 3 weeks). The results show increased growth delay for the combination of irradiation + 5E1. D) Mean weight measurements for the various treated mice in panels B and C indicating significant toxicity (requiring euthanasia of some mice) in the groups treated with combination rads + cisplatin. No weight loss was observed in the mice treated with 5E1.

Supported by

Irradiation set-up Photo of image-guided irradiator (X-Rad 225Cx, Precision X-ray, North Branford, CT, USA). The X-ray tube in this unit is mounted on a rotating C arm with a flat panel detector opposite for image-guided set-up.

S c r e e n s h o t o f treatment planning for the orthotopic cervix xenografts showing the beam arrangement of 8-equally spaced beams.

A B

Tumor size

Lymph node metastasis

N=5 mice/grp. (NOD/SCID mice)

A Rag2 -/- /gammaT null mice (4 mice /gp)

C

B D

NOD/Rag1-/- /gammaT null (NRG) mice (4-5 mice/gp)