Interactions between neuroblastoma and the immune system : cellular pathways and mediators

Neuroblastoma (NB) is an embryonal tumor of early childhood arising in tissues of the sympathetic nervous system, such as the adrenal gland and paraspinal ganglia. It is the most common extra-cranial solid tumor of childhood, and 10-20 children are diagnosed with NB each year in Sweden. The overall survival rate is about 70%, but 50% of the children in the high-risk group succumb in spite of intense multimodal therapy. This warrants the search for alternative treatment strategies. One upcoming treatment option is immunotherapy, which represents a specific treatment modality with the possibility of minimizing long-term side effects in survivors.

Cellular therapies for NB have previously been discouraged due to the notion that NB is a tumor of low immunogenicity. This thesis demonstrates that differentiating agents alter the immune phenotype of primary NB tumors and cell lines such as to enhance the expression of classical HLA molecules and the adhesion molecule ICAM-1. This was paralleled by an increased ability of differentiated NB cells to bind granzyme B at the cell surface and translated into enhanced killing by natural killer (NK) cells and T-cells. These results argue in favor of differentiation and cellular immunotherapy as a combined auxiliary approach for NB patients (paper I). Furthermore, the work presented in this thesis demonstrates that tumor-non-specific activated cytotoxic T lymphocytes (CTLs) release effector molecules which facilitate immune-mediated recognition of NB. Effector molecules from CTLs upregulated HLA class I, ICAM-1 and Fas at the cell surface and restored the expression and activity of caspase-8 in primary NB tumors and cell lines. This rendered NB cells more susceptible to death receptor-mediated killing (paper II).

This thesis also demonstrates that primary human NB samples, representing all genetical subtypes, harbor tumor-infiltrating T-cells which proliferate in situ. Tumor-infiltrating lymphocytes were preferentially CD8+, expressed high levels of the activation marker CD25 and exhibited a phenotype of memory cells. Autologous peripheral blood lymphocytes were exposed to tumor cells in vitro and their production of IFN-γ and TNF-α was increased, while an activated phenotype was obtained. This indicates that human NB cells do not prevent the generation of active T-cell responses (paper III). In the transgenic TH-MYCN mouse model of NB, tumor-associated inflammation was investigated and NB tumor progression was shown to be paralleled by a gradual suppression of intratumoral T-cell responses in favor of immature cells of the innate immune system. Anti-inflammatory treatment with low-dose aspirin displayed a promising efficacy in delaying tumor outgrowth with a concomitant abrogation of an inflammatory switch (paper IV).

Taken together, the work presented in this thesis demonstrates that NB can serve as a proper target for cellular immunotherapy. It argues for an early implementation of immunotherapy in clinical protocols, where differentiating agents and/or the attraction of activated CTLs to the NB microenvironment could enhance immune-mediated tumor recognition.

List of scientific papers

I. Lena-Maria Carlson, Sven Påhlman, Anna De Geer, Per Kogner and Jelena Levitskaya. Differentiation induced by physiological and pharmacological stimuli leads to increased antigenicity of human neuroblastoma cells. Cell Research. 2008, 18:398-411.
https://doi.org/10.1038/cr.2008.27

II. Anna De Geer, Lena-Maria Carlson, Per Kogner and Jelena Levitskaya. Soluble factors released by activated cytotoxic T lymphocytes interfere with death receptor pathways in neuroblastoma. Cancer Immunology, Immunotherapy. 2008, 57:731-743.
https://doi.org/10.1007/s00262-007-0412-2

III. Lena-Maria Carlson*, Anna De Geer*, Baldur Sveinbjörnsson, Abiel Orrego, Tommy Martinsson, Per Kogner and Jelena Levitskaya. Human neuroblastoma microenvironment supports T-cell activation in tumor-associated lymphocytes. [Manuscript]

IV. Lena-Maria Carlson, Agnes Rasmuson, Lova Segerström, Baldur Sveinbjörnsson and Per Kogner. Progressive tumor-associated inflammation as a potential therapeutic target in neuroblastoma. [Manuscript]