Additional file 2: Figure S2. of Enhanced recruitment of genetically modified CX3CR1-positive human T cells into Fractalkine/CX3CL1 expressing tumors: importance of the chemokine gradient

Transduction of human T lymphocytes with eGFP/CX3CR1-eGFP gene. (A) Population of CD3+ and of CD4+/CD8+ T cells post retroviral transduction of eGFP/CX3CR1 or -eGFP gene after 3 days of in vitro culture with hIL-2 medium. (JPG 1522 kb

Additional file 3: Figure S3. of Enhanced recruitment of genetically modified CX3CR1-positive human T cells into Fractalkine/CX3CL1 expressing tumors: importance of the chemokine gradient

Lymphocyte infiltration in the lungs of tumor-bearing mice after adoptive transfer. Flow cytometry analysis of T lymphocytes entrapped in the lungs of mice receiving adoptive transfer of CX3CR1-T cells or GFP-T cells (3 mice per group). (A) Proportion of CD3+ T lymphocytes among CD45+ cells in the disaggregated lungs from mice bearing RKO-Mock or RKO-CX3CL1 tumors. (B) Proportion of CD3+ T lymphocytes among CD45+ cells in the disaggregated lungs from mice bearing NCI-H630 tumors. (JPG 1661 kb

Kaplan—Meier curves showing disease-specific survival (DSS) after resection with curative intent for colorectal cancer according to plasma values of IL-8, PTX-3, VEGF and the immune score.

<p>The ROC threshold values of IL-8, PTX3 and VEGF were calculated in the whole cohort (6.79 pg/ml, 31.97pg/ml, 6.29pg/ml, respectively) and were used as a cutoff to select high and low IL-8, PTX-3 and VEGF in colorectal cancer patients. Low immune score was defined by combined lowIL-8-lowVEGF-lowPTX3 values. High immune score was defined by combined highIL-8-highVEGF-highPTX3 values. Intermediate score was defined by any other value. Values above the ROC cutoff were associated with worse patient’s survival for CXCL8 (P = 0.008), PTX3 (P = 0.001), VEGF (P = 0.005) and the immune score (P<0.001), compared to those below ROC cutoff.</p

Continuous values of IL-1, IL-6, IL-10, TNF-a, CCL2, CXCL8, VEGF and the acute phase protein Pentraxin-3 in the plasma of colorectal cancers according to the occurrence of postsurgical relapse.

<p>The amount of IL-1 (A), IL-6 (B), IL-10 (C), TNF-a (D), IL-8 (F), VEGF (G) significantly increased in patients who experienced disease relapse, while the acute phase protein Pentraxin-3 (H) showed a tendency to associate with worse prognosis. CCL2 (E) levels were not associated with disease relapse.</p

DataSheet_1_Polymeric nanocapsules loaded with poly(I:C) and resiquimod to reprogram tumor-associated macrophages for the treatment of solid tumors.pdf

BackgroundIn the tumor microenvironment (TME), tumor-associated macrophages (TAMs) play a key immunosuppressive role that limits the ability of the immune system to fight cancer. Toll-like receptors (TLRs) ligands, such as poly(I:C) or resiquimod (R848) are able to reprogram TAMs towards M1-like antitumor effector cells. The objective of our work has been to develop and evaluate polymeric nanocapsules (NCs) loaded with poly(I:C)+R848, to improve drug stability and systemic toxicity, and evaluate their targeting and therapeutic activity towards TAMs in the TME of solid tumors.MethodsNCs were developed by the solvent displacement and layer-by-layer methodologies and characterized by dynamic light scattering and nanoparticle tracking analysis. Hyaluronic acid (HA) was chemically functionalized with mannose for the coating of the NCs to target TAMs. NCs loaded with TLR ligands were evaluated in vitro for toxicity and immunostimulatory activity by Alamar Blue, ELISA and flow cytometry, using primary human monocyte-derived macrophages. For in vivo experiments, the CMT167 lung cancer model and the MN/MCA1 fibrosarcoma model metastasizing to lungs were used; tumor-infiltrating leukocytes were evaluated by flow cytometry and multispectral immunophenotyping.ResultsWe have developed polymeric NCs loaded with poly(I:C)+R848. Among a series of 5 lead prototypes, protamine-NCs were selected based on their physicochemical properties (size, charge, stability) and in vitro characterization, showing good biocompatibility on primary macrophages and ability to stimulate their production of T-cell attracting chemokines (CXCL10, CCL5) and to induce M1-like macrophages cytotoxicity towards tumor cells. In mouse tumor models, the intratumoral injection of poly(I:C)+R848-protamine-NCs significantly prevented tumor growth and lung metastasis. In an orthotopic murine lung cancer model, the intravenous administration of poly(I:C)+R848-prot-NCs, coated with an additional layer of HA-mannose to improve TAM-targeting, resulted in good antitumoral efficacy with no apparent systemic toxicity. While no significant alterations were observed in T cell numbers (CD8, CD4 or Treg), TAM-reprogramming in treated mice was confirmed by the relative decrease of interstitial versus alveolar macrophages, having higher CD86 expression but lower CD206 and Arg1 expression in the same cells, in treated mice.ConclusionMannose-HA-protamine-NCs loaded with poly(I:C)+R848 successfully reprogram TAMs in vivo, and reduce tumor progression and metastasis spread in mouse tumors.</p