In order to achieve the proposed objectives, a combination of immunohistochemistry, multiparametric flow cytometry, gene expression analysis and in vivo experiments, among other studies, were performed.
Aim 1 involved the characterization of the tumor immune infiltrate into the two distinct TNBC subtypes, defined by their level of p-P70S6K. Refined image analyses were performed from tumor samples of 341 TNBC patients. TIL infiltration was evaluated and a reduced stromal TILs (sTILs) percentage was found in those patients exhibiting hyperactivation of the kinase. Further classification of tumor lymphocytes showed that B cell abundance was lower in high p-P70S6K samples. Additionally, a decreased CD8/CD4 ratio was found in high p-P70S6K samples. Moreover, positive correlations were found between the levels of p-P70S6K and CD4 and Foxp3 expression, and CD8/CD4 and CD8/Foxp3 ratios. Overall, these analyses allowed knowing the lymphocytes dysregulated in TNBC patients with high/low p-P70S6K tumor levels.
Aim 2 explored the role of P70S6K in tumor immunogenicity and T cell responses in TNBC. Initial part of this objective was focused on assessing the potential dual role of P70S6K on tumor and TIL compartments in TNBC. First, to know the mechanisms by which hyperactivation of tumor P70S6K could influence tumor immunogenicity, several analyses were completed by in vitro blockade of P70S6K activity in different TNBC cell lines. These analyses included the study of: molecules involved in presentation of tumor antigens to T cells, expression of immune checkpoint inhibitors or release of molecules supporting in T cell recruitment into the tumor. A heterogenous response was obtained from these experiments depending on cell line and inhibitor used. Second, the effect of P70S6K inhibition on T cells was studied. Ex vivo cultures of T cells from tumor or spleen were performed under basal or stimulated conditions and in the presence/absence of inhibitors of P70S6K. Results suggested that inhibition of P70S6K activity has a negative effect on anti-tumoral T cell responses, while promoting suppressive immune responses.
The second part of Aim 2 included the evaluation of the therapeutic effect of P70S6K blockade in preclinical models of TNBC and its potential synergies with current immunotherapies. Pharmacological inhibition of P70S6K was tested in vivo in different mouse models of TNBC. Results revealed that inhibition of P70S6K activity significantly delayed tumor growth and increase survival of mice wearing tumors over-expressing p-P70S6K.
Altogether, these in vitro and in vivo experiments laid the foundation for forthcoming elucidation of the role of P70S6K on tumor immunogenicity and T cell responses in TNBC.
Throughout the length of the project, results found in the above-mentioned experiments were available to the scientific community and public by different channels. Public had the opportunity to know the project in different outreach activities, such as the European Researchers´ Night or the XI Feria Madrid es Ciencia 2022. Moreover, the project has been published in a report entitled ´Breast cancer: an aggressive variant triggers a hunt for cures´ (published in Horizon, The EU Research & Innovation Magazine on 9th December 2022 and in EL PAIS on 20th December 2022; and posted at CNIO twitter). Likewise, data was presented to scientific colleagues through diverse ways: labmeeting seminars, CNIO Progress Report seminars, Clinical Research Programme seminars and other internal seminars. Importantly, data from this project was published in an Open Access peer-review article entitled ´Tumor P70S6K hyperactivation is inversely associated with tumor-infiltrating lymphocytes in triple-negative breast cancer´ (Clin Transl Oncol. 2022. doi: 10.1007/s12094-022-03006-3).
