Access Type

Open Access Dissertation

Date of Award

January 2013

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Cancer Biology

First Advisor

Wei-zen Wei

Abstract

Background and Objectives: Percutaneous cryoablation is a minimally invasive procedure for tumor debulking, which has the potential to initiate or amplify tumor immunity through the release of tumor-associated antigens and endogenous danger signals. However, enhanced immunity is rarely observed in treated patients, suggesting the need for mechanistic analysis. The goal is to determine how cryoablation affects tumor specific immunity and if the response can be improved through exogenous TLR9 stimulation.

Methodology: We evaluated anti-Her2/neu immunity following cryoablation in wt BALB/c and tolerant NeuT mice inoculated with neu or Her2 expressing mammary tumors TUBO and D2F2/E2 respectively. Mice were treated with cryoablation, tumor excision, sham surgery, and/or 100 micrograms peritumoral (p.t.) CpG ODN. NeuT mice received vaccination with plasmid DNA encoding neu/Her2 and GM-CSF to induce an initial response. Specific IgG antibody (Ab) subclasses and T-cell responses were assessed using flow cytometry and IFN gamma ELISPOT assays respectively. Inflammatory transcript and protein levels from stimulated tumor draining lymph nodes (TDLN) were quantified using qPCR and magnetic bead multiplexing respectively. Phenotyping of peripheral blood leukocytes and TDLN was performed using flow cytometry.

Results: Cryoablation of Ab-sensitive TUBO induced anti-neu Ab that protected ~65% of wt mice from tumor re-challenge which increased to ~95% when p.t. CpG was used in combination therapy. Cryoablation of D2F2/E2 protected ~80% of mice if rechallenge was performed after the ablated tumor had been cleared (41 d). However, rechallenge 2 weeks after cryoablation resulted in accelerated growth relative to surgical incision. In vaccinated tolerant NeuT mice, protection was not amplified after cryoablation, even with the addition of CpG. However, tumor excision provided significantly greater tumor protection. Cryoablation primarily induced anti-neu IgG1 relative to IgG2a, which was inversed with the addition of CpG. Cryoablation elevated many inflammatory transcripts, with the most significantly elevated transcripts indicative of a Th2 phenotype (Il10 and Il4) and suppression (Foxp3 and Tgfb). Recurrences of tumors treated with cryoablation occurred in 26-29% of WT mice which was significantly decreased to 0% with the addition of CpG.

Conclusions: These findings suggest cryoablation induces a Th2 dominant response, which may be detrimental if residual disease is present. To promote the shift to a Th1 phenotype, which is associated with greater anti-tumor activity, CpG was used in combination with cryoablation. This led to significantly elevated IgG2a/IgG1 relative to cryoablation alone and greater tumor protection in wt mice, suggesting Th1 activation. Furthermore, the addition of CpG elevated IFN gamma responses above that of cryoablation alone, which appeared to primarily be localized to the treated region. Cryoablation can be an effective tool for both tumor debulking and immune priming if Th1-promoting adjuvants are used in combination therapy.

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