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mRNA-based vaccines synergize with radiation therapy to eradicate established tumors.

Fotin-Mleczek M, Zanzinger K, Heidenreich R, Lorenz C, Kowalczyk A, Kallen KJ, Huber SM - Radiat Oncol (2014)

Bottom Line: In both tumor models we demonstrated that a combination of mRNA-based immunotherapy with radiation results in a strong synergistic anti-tumor effect.Genes associated with antigen presentation, infiltration of immune cells, adhesion, and activation of the innate immune system were upregulated.Our data provide a scientific rationale for combining immunotherapy with radiation and provide a basis for the development of more potent anti-cancer therapies.

View Article: PubMed Central - PubMed

Affiliation: CureVac GmbH, CureVac GmbH, Paul-Ehrlich-Str, 15, Tübingen 72076, Germany. mf@curevac.com.

ABSTRACT

Background: The eradication of large, established tumors by active immunotherapy is a major challenge because of the numerous cancer evasion mechanisms that exist. This study aimed to establish a novel combination therapy consisting of messenger RNA (mRNA)-based cancer vaccines and radiation, which would facilitate the effective treatment of established tumors with aggressive growth kinetics.

Methods: The combination of a tumor-specific mRNA-based vaccination with radiation was tested in two syngeneic tumor models, a highly immunogenic E.G7-OVA and a low immunogenic Lewis lung cancer (LLC). The molecular mechanism induced by the combination therapy was evaluated via gene expression arrays as well as flow cytometry analyses of tumor infiltrating cells.

Results: In both tumor models we demonstrated that a combination of mRNA-based immunotherapy with radiation results in a strong synergistic anti-tumor effect. This was manifested as either complete tumor eradication or delay in tumor growth. Gene expression analysis of mouse tumors revealed a variety of substantial changes at the tumor site following radiation. Genes associated with antigen presentation, infiltration of immune cells, adhesion, and activation of the innate immune system were upregulated. A combination of radiation and immunotherapy induced significant downregulation of tumor associated factors and upregulation of tumor suppressors. Moreover, combination therapy significantly increased CD4+, CD8+ and NKT cell infiltration of mouse tumors.

Conclusion: Our data provide a scientific rationale for combining immunotherapy with radiation and provide a basis for the development of more potent anti-cancer therapies.

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Related in: MedlinePlus

Infiltration of immune cells into the large established immunosuppressive LLC tumors is improved after combination therapy. C57BL/6 mice (n = 4 per group) were challenged subcutaneously on the right hind limb with 5 × 105 3LL-GFP cells. 18 days after tumor challenge mice were treated with local radiation alone (36 Gy total, divided into 3 equal fractions on 3 consecutive days) (RTX alone), vaccination alone (32 μg, twice a week, started at day 18) (RNA alone) or with radioimmunotherapy (with first vaccination given on day 19) (RNA + RTX). Untreated mice served as a control (No therapy). On day 25 tumors were excised, homogenized and immune cell infiltration analyzed by flow cytometry. (A) MHC class I expression was analyzed on single cell suspension of untreated- and irradiated tumor cells. (B) CD45.2+ host-derived immune cells were characterized by cell surface staining of several lineage markers and the ratio of different CD45.2+ immune cell subpopulations to CD45.2- tumor cells is shown as median of four animals per group. * p < 0.05. Presented data show representative results of two independent analyses.
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Fig4: Infiltration of immune cells into the large established immunosuppressive LLC tumors is improved after combination therapy. C57BL/6 mice (n = 4 per group) were challenged subcutaneously on the right hind limb with 5 × 105 3LL-GFP cells. 18 days after tumor challenge mice were treated with local radiation alone (36 Gy total, divided into 3 equal fractions on 3 consecutive days) (RTX alone), vaccination alone (32 μg, twice a week, started at day 18) (RNA alone) or with radioimmunotherapy (with first vaccination given on day 19) (RNA + RTX). Untreated mice served as a control (No therapy). On day 25 tumors were excised, homogenized and immune cell infiltration analyzed by flow cytometry. (A) MHC class I expression was analyzed on single cell suspension of untreated- and irradiated tumor cells. (B) CD45.2+ host-derived immune cells were characterized by cell surface staining of several lineage markers and the ratio of different CD45.2+ immune cell subpopulations to CD45.2- tumor cells is shown as median of four animals per group. * p < 0.05. Presented data show representative results of two independent analyses.

Mentions: Gene expression analysis revealed a number of substantial and specific changes at the tumor sites following different therapies. Next, we asked whether these changes are reflected in the cellular composition of the tumor tissue. To obtain this information, we analyzed LLC tumor infiltrates by flow cytometry. LLC tumor cells were transplanted subcutaneously into the right limb and mice were left untreated until the tumor reached a volume of about 50 mm3. Tumors of the radiation (RTX alone) and the combined radioimmunotherapy (RNA + RTX) groups were irradiated with a total dose of 36 Gy, administered in 3 fractions of 12 Gy each on three consecutive days. Mice in the immunotherapy (RNA alone) and the radioimmunotherapy groups received two vaccinations per week. Untreated mice served as controls (No therapy). On day 25, tumors were isolated and the cellular composition was analyzed. At this time point, tumors did not differ significantly in volume (median volume was about 150-200 mm3). As shown in Figure 4A, irradiation of LLC tumors led to a strong upregulation of the MHC class I molecule. Additionally, the cellular composition of the tumor microenvironment was dramatically changed after irradiation and combination therapy compared to the untreated control tumors. As shown in Figure 4B when different populations of the tumor infiltrating immune cells were analyzed, we observed that tumor radiation induced a strong increase of myeloid and dendritic cells which are associated with activation of innate immune system. This was also the case for the combination therapy. Importantly, compared to control mice tumors from mice treated with combined immunoradiotherapy exhibited statistically significant increase in infiltration of CD4+, CD8+ T cells and NKT cells which are associated with the adaptive immune system. Despite the late onset of the immunotherapy also after vaccination alone improved infiltration of CD8+ T cells (fold change 1.4) and CD4+ T cells (fold change 0.6) could be observed suggesting beneficial effects of the immunotherapy even with the high tumor burden. The frequency of B cells was very low and levels did not change after the various treatments (data not shown). Taken together, our results show that local radiation of large established tumors results in cellular changes in the tumor microenvironment, making the tumor more accessible to mRNA-based immunotherapy.Figure 4


mRNA-based vaccines synergize with radiation therapy to eradicate established tumors.

Fotin-Mleczek M, Zanzinger K, Heidenreich R, Lorenz C, Kowalczyk A, Kallen KJ, Huber SM - Radiat Oncol (2014)

Infiltration of immune cells into the large established immunosuppressive LLC tumors is improved after combination therapy. C57BL/6 mice (n = 4 per group) were challenged subcutaneously on the right hind limb with 5 × 105 3LL-GFP cells. 18 days after tumor challenge mice were treated with local radiation alone (36 Gy total, divided into 3 equal fractions on 3 consecutive days) (RTX alone), vaccination alone (32 μg, twice a week, started at day 18) (RNA alone) or with radioimmunotherapy (with first vaccination given on day 19) (RNA + RTX). Untreated mice served as a control (No therapy). On day 25 tumors were excised, homogenized and immune cell infiltration analyzed by flow cytometry. (A) MHC class I expression was analyzed on single cell suspension of untreated- and irradiated tumor cells. (B) CD45.2+ host-derived immune cells were characterized by cell surface staining of several lineage markers and the ratio of different CD45.2+ immune cell subpopulations to CD45.2- tumor cells is shown as median of four animals per group. * p < 0.05. Presented data show representative results of two independent analyses.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig4: Infiltration of immune cells into the large established immunosuppressive LLC tumors is improved after combination therapy. C57BL/6 mice (n = 4 per group) were challenged subcutaneously on the right hind limb with 5 × 105 3LL-GFP cells. 18 days after tumor challenge mice were treated with local radiation alone (36 Gy total, divided into 3 equal fractions on 3 consecutive days) (RTX alone), vaccination alone (32 μg, twice a week, started at day 18) (RNA alone) or with radioimmunotherapy (with first vaccination given on day 19) (RNA + RTX). Untreated mice served as a control (No therapy). On day 25 tumors were excised, homogenized and immune cell infiltration analyzed by flow cytometry. (A) MHC class I expression was analyzed on single cell suspension of untreated- and irradiated tumor cells. (B) CD45.2+ host-derived immune cells were characterized by cell surface staining of several lineage markers and the ratio of different CD45.2+ immune cell subpopulations to CD45.2- tumor cells is shown as median of four animals per group. * p < 0.05. Presented data show representative results of two independent analyses.
Mentions: Gene expression analysis revealed a number of substantial and specific changes at the tumor sites following different therapies. Next, we asked whether these changes are reflected in the cellular composition of the tumor tissue. To obtain this information, we analyzed LLC tumor infiltrates by flow cytometry. LLC tumor cells were transplanted subcutaneously into the right limb and mice were left untreated until the tumor reached a volume of about 50 mm3. Tumors of the radiation (RTX alone) and the combined radioimmunotherapy (RNA + RTX) groups were irradiated with a total dose of 36 Gy, administered in 3 fractions of 12 Gy each on three consecutive days. Mice in the immunotherapy (RNA alone) and the radioimmunotherapy groups received two vaccinations per week. Untreated mice served as controls (No therapy). On day 25, tumors were isolated and the cellular composition was analyzed. At this time point, tumors did not differ significantly in volume (median volume was about 150-200 mm3). As shown in Figure 4A, irradiation of LLC tumors led to a strong upregulation of the MHC class I molecule. Additionally, the cellular composition of the tumor microenvironment was dramatically changed after irradiation and combination therapy compared to the untreated control tumors. As shown in Figure 4B when different populations of the tumor infiltrating immune cells were analyzed, we observed that tumor radiation induced a strong increase of myeloid and dendritic cells which are associated with activation of innate immune system. This was also the case for the combination therapy. Importantly, compared to control mice tumors from mice treated with combined immunoradiotherapy exhibited statistically significant increase in infiltration of CD4+, CD8+ T cells and NKT cells which are associated with the adaptive immune system. Despite the late onset of the immunotherapy also after vaccination alone improved infiltration of CD8+ T cells (fold change 1.4) and CD4+ T cells (fold change 0.6) could be observed suggesting beneficial effects of the immunotherapy even with the high tumor burden. The frequency of B cells was very low and levels did not change after the various treatments (data not shown). Taken together, our results show that local radiation of large established tumors results in cellular changes in the tumor microenvironment, making the tumor more accessible to mRNA-based immunotherapy.Figure 4

Bottom Line: In both tumor models we demonstrated that a combination of mRNA-based immunotherapy with radiation results in a strong synergistic anti-tumor effect.Genes associated with antigen presentation, infiltration of immune cells, adhesion, and activation of the innate immune system were upregulated.Our data provide a scientific rationale for combining immunotherapy with radiation and provide a basis for the development of more potent anti-cancer therapies.

View Article: PubMed Central - PubMed

Affiliation: CureVac GmbH, CureVac GmbH, Paul-Ehrlich-Str, 15, Tübingen 72076, Germany. mf@curevac.com.

ABSTRACT

Background: The eradication of large, established tumors by active immunotherapy is a major challenge because of the numerous cancer evasion mechanisms that exist. This study aimed to establish a novel combination therapy consisting of messenger RNA (mRNA)-based cancer vaccines and radiation, which would facilitate the effective treatment of established tumors with aggressive growth kinetics.

Methods: The combination of a tumor-specific mRNA-based vaccination with radiation was tested in two syngeneic tumor models, a highly immunogenic E.G7-OVA and a low immunogenic Lewis lung cancer (LLC). The molecular mechanism induced by the combination therapy was evaluated via gene expression arrays as well as flow cytometry analyses of tumor infiltrating cells.

Results: In both tumor models we demonstrated that a combination of mRNA-based immunotherapy with radiation results in a strong synergistic anti-tumor effect. This was manifested as either complete tumor eradication or delay in tumor growth. Gene expression analysis of mouse tumors revealed a variety of substantial changes at the tumor site following radiation. Genes associated with antigen presentation, infiltration of immune cells, adhesion, and activation of the innate immune system were upregulated. A combination of radiation and immunotherapy induced significant downregulation of tumor associated factors and upregulation of tumor suppressors. Moreover, combination therapy significantly increased CD4+, CD8+ and NKT cell infiltration of mouse tumors.

Conclusion: Our data provide a scientific rationale for combining immunotherapy with radiation and provide a basis for the development of more potent anti-cancer therapies.

Show MeSH
Related in: MedlinePlus