A Single-Cell Atlas Of The Effect Of Chemotherapeutics Over Intratumoral Immune Cells Reveals That Combining An Alkylating Agent And A Vinca Alkaloid Can Activate Antigen Presenting Cells And Increase Tcf1+Stem-Like Cd8+T-Cells, Thus Improving Anti-Pd-1 Efficacy In Triple Negative Breast Cancer And Lymphoma.

Abstract Checkpoint inhibitors (CIs) instigate anticancer immunity, albeit only in a fraction of patients. Triple negative breast cancer (TNBC) is among the most aggressive and lethal BC types, and currently available therapies have an unsatisfactory clinical impact. The association of CIs with chemotherapy had encouraging results in randomized TNBC trials, but so far there has been no clear evidence of what should be considered the best combinatorial therapy. In B-cell non-Hodgkin's lympoma (NHL), CIs' clinical activity has been so far unsatisfactory. We designed the current study to define what could be considered the most efficient combinatorial regimen of chemotherapy plus CI in TNBC and NHL. We investigated different options including platinum (P), doxorubicin (D), taxol (T), vinorelbine (V), and cyclophosphamide ( C), i.e. the most effective chemotherapy drugs for these diseases. Capecitabine was not considered as found not to be effective in combination with CIs. As TNBC patients have a large intratumoral immune cell heterogeneity, we investigated two immune competent TNBC models: 4T1 with a predominant lymphoid intratumoral infiltrate and EMT6 with a predominant myeloid infiltrate. A C57BL/6 Eμ-myc transgenic NHL model, with a predominant lymphatic dissemination pattern, was also studied. We found that 1) Intermittent, medium-dosage C (C140), was more effective than other combinatorial regimens including the CI anti-PD1 plus low-dose C, or regular-dose T, D, or P. The association of V and anti-PD1 further increased the preclinical efficacy of C140; 2) both CD3+CD4+ and CD3+CD8+ T cells (at variance with other subsets of NK and myeloid cells) were needed to control local and metastatic neoplastic growth in mice treated with C140, V and anti-PD1. 3) The combinatorial therapy including C140, V and anti-PD1 was the most efficient in activating antigen presenting cells (APCs), and the investigation of TCR repertoire indicated that this therapy generated a significantly larger clonal expansion of both CD3+CD4+ and CD3+CD8+ T cells. 4) Single-cell transcriptome analysis of >50,000 intratumoural immune cells showed after C140, V and anti-PD1 therapy a gene signature suggestive of a change resulting from exposure to a mitogen, ligand, or an antigen for which it is specific, as well as APC-to-T-cell adhesion. This transcriptional program also increased intratumoural tcf1+ stem-like CD8+ T-cells and altered the balance between terminally and progenitor exhausted T-cells, favoring the latter. These data support the clinical investigation of this therapy in TNBC and NHL, and we are currently investigating the related transcriptional mechanisms inducing APC activation and T cell rewiring. Citation Format: Paolo Falvo, Stefania Orecchioni, Roman Hillje, Alessandro Raveane, Giulia Mitola, Patrizia Mancuso, Chiara Camisaschi, Lucilla Luzi, PierGiuseppe Pelicci, Francesco Bertolini. A single-cell atlas of the effect of chemotherapeutics over intratumoral immune cells reveals that combining an alkylating agent and a vinca alkaloid can activate antigen presenting cells and increase tcf1+ stem-like CD8+ T-cells, thus improving anti-PD-1 efficacy in triple negative breast cancer and lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1653.