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Photon versus carbon ion radiotherapy – combining radiotherapy with immune checkpoint blockade in the EO771 breast cancer model

Hartmann, Laura

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Abstract

Carbon ion radiation is characterized by favorable physical and biological properties when compared to classical photon radiation. While photon radiation has been shown to act as an in situ vaccine inducing immunological cell death, little is known about the immunomodulatory capacity of carbon ion radiation. Thus, the aim of this study was to compare the immunomodulatory potential of photon versus carbon ion radiation.

In the first part of this thesis, cytotoxic and immunomodulatory effects of photon and carbon ion radiation on the murine breast cancer cell line EO771 were analyzed in vitro. In clonogenic survival assays, biologically equivalent doses of 0.12, 1.11, 3.08, 8.0 Gy carbon ions corresponding to 1, 3, 5, and 10 Gy photons in the EO771 cell line were defined. Applying these doses, photon and carbon ion radiation induced comparable radiogenic effects in vitro. Thus, irradiation elicited a dose-dependent cytotoxicity as increasing doses reduced proliferation, initiated a transient G2/M cell cycle arrest, and induced apoptosis and cell death. Likewise, a dose-dependent enhanced expression of the immunomodulatory molecules PD L1, CD73, and MHC class I molecules (H2-Kb and H2 Db), both on the mRNA and protein level, as well as increased secretion of the danger signal HMGB1 were observed. Furthermore, both radiation types made tumor cells more sensitive to CTL-mediated killing in a dose-dependent manner, while the CTLs’ IFN γ response levels remained constant.

In the second part of this thesis, a bilateral tumor model was established as radiotherapy (RT) of a primary tumor can induce antitumor immune responses that might even induce the regression of distant metastases (abscopal effect). In fact, therapeutic effects on both local (irradiated, tumor #1) and distant (non-irradiated, tumor #2) tumors could be monitored in this tumor model. As abscopal effects only rarely occur, most likely due to radiation-induced immunosuppression, RT was combined with immune checkpoint blockade against PD L1 or CTLA-4. Both photon and carbon ion RT alone could control local tumor growth to limited extent, while growth of distant tumors was not affected. Interestingly, photon RT appeared slightly superior to carbon ion RT when used as monotherapy. Combination of RT with anti PD L1 antibodies further enhanced antitumor effects on tumor #1, but had only a marginal impact on tumor #2. Remarkably, RT plus anti-CTLA-4 checkpoint blockade showed superior therapeutic efficacies on both tumor #1 and tumor #2 leading to complete responses in up to 80% of mice. Using combination therapy with anti-CTLA-4 antibodies, a side-by-side comparison of photon vs. carbon ion RT proved to be equally effective.

In tumor #1, photon and carbon ion RT plus anti-CTLA-4 therapy was associated with decreased frequencies of Tregs and CD4+CTLA-4+ T cells, decreased infiltration by CD4+ and CD8+ T cells expressing the immune checkpoint molecules PD-1 and LAG-3, and high numbers of CD8+CD73+ T cells. Overall, these observations point towards an activated and functional phenotype of T cells in the tumor microenvironment. In tumor #2, combination therapy resulted in decreased Treg and increased CD8+ T cell infiltration, however due to high variations between single tumors within a group, no significant differences were observed in expression levels of the other markers investigated. Finally, 100% of mice showing complete responses after photon RT plus anti CTLA-4 therapy rejected a secondary tumor engraftment. Moreover, splenocytes of these mice released INF-γ in response to EO771 tumor cells, but not to other tumor cell lines, demonstrating the establishment of tumor-specific memory immune responses. Taken together, this thesis shows that photon and carbon ion radiation induce comparable cytotoxic and immunomodulatory effects when using biologically equivalent doses. Due to the outstanding antitumor effects on local and distant tumors, both radiation types provide great potential for clinical cancer management when applied in combination with immune checkpoint blockade. However, based on its favorable physical and biological properties allowing precise irradiation of tumors, while sparing normal tissue, carbon ion RT appears as preferable treatment option.

Item Type: Dissertation
Supervisor: Müller, Prof. Dr. Martin
Place of Publication: Heidelberg
Date of thesis defense: 19 May 2021
Date Deposited: 16 Jun 2021 10:45
Date: 2022
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 570 Life sciences
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