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PDF file - 193K, Representative flow cytometry analysis of a CIK cell culture generated from PBMC from mMel patients.

Purpose: We investigate the unknown tumor-killing activity of cytokine-induced killer (CIK) cells against autologous metastatic melanoma and the elusive subset of putative cancer stem cells (mCSC).Experimental Design: We developed a preclinical autologous model using same patient-generated CIK cells and tumor targets to consider the unique biology of each patient/tumor pairing. In primary tumor cell cultures, we visualized and immunophenotypically defined a putative mCSC subset using a novel gene transfer strategy that exploited their exclusive ability to activate the promoter of stemness gene Oct4.Results: The CIK cells from 10 patients with metastatic melanoma were successfully expanded (median, 23-fold; range, 11–117). Primary tumor cell cultures established and characterized from the same patients were used as autologous targets. Patient-derived CIK cells efficiently killed autologous metastatic melanoma [up to 71% specific killing (n = 26)]. CIK cells were active in vivo against autologous melanoma, resulting in delayed tumor growth, increased necrotic areas, and lymphocyte infiltration at tumor sites. The metastatic melanoma cultures presented an average of 11.5% ± 2.5% putative mCSCs, which was assessed by Oct4 promoter activity and stemness marker expression (Oct4, ABCG2, ALDH, MITF). Expression was confirmed on mCSC target molecules recognized by CIK cells (MIC A/B; ULBPs). CIK tumor killing activity against mCSCs was intense (up to 71%, n = 4) and comparable with results reported against differentiated metastatic melanoma cells (P = 0.8).Conclusions: For the first time, the intense killing activity of CIK cells against autologous metastatic melanoma, including mCSCs, has been shown. These findings move clinical investigation of a new immunotherapy for metastatic melanoma, including mCSCs, closer. Clin Cancer Res; 19(16); 4347–58. ©2013 AACR.

PDF file - 147K, Representative flow cytometry analysis of melanoma primary cell cultures. assessment of CD117 (aka c-kit) and MCSP; c) assessment of MIC A/B and ULBP2; d) assessment of ULBP1 and ULBP3 expression; e) expression assessment of CD34 and VEGFR1 expression; f) assessment of intracellular Oct4 expression in fixed and permeabilized cells; g) assessment of ABCG2 expression; h) assessment of ALDH activity in cells stained with FITC ALDH substrate; i) assessment of MITF negativity in fixed and permeabilized cells stained with a primary specific antibody unconjugated and a PE conjugated secondary antibody; k) assessment of CD117 (also known as c-kit) and MCSP in mMel6, notably highly positive for CD117 expression.

PDF file - 143K, Primary melanoma cell targets.

Supplementary Figure S2: Immunophenotype of patient-derived CIK cells and CIK cell in vitro activity

DNA extraction and Sanger Sequencing In vivo Limiting Dilution Assay Cell growth inhibition assay Analysis of LV.Oct4.eGFP integration in eGFP positive and negative cell fractions

PDF file - 64K

PDF file - 122K, Representative cell sorting plots of LV-oct4.eGFP transduced cells.

PDF file - 117K, Expression level of ABCG2 (a), NKG2D ligands MIC A/B (b) and ULBP2 (c) in LV-oct4-eGFP transduced cells.

PDF file - 81K, Representative flow cytometry analysis of melanoma primary transduced cells.

Supplementary Table 1: Patients Characteristics Supplementary Table 2: Comparative transduction of melanoma cell cultures

Supplementary Figure S5:Tumor infiltration by CIK cells

Purpose: The MHC-unrestricted activity of cytokine-induced killer (CIK) cells against chemo-surviving melanoma cancer stem cells (mCSC) was explored, as CSCs are considered responsible for chemoresistance and relapses.Experimental Design: Putative mCSCs were visualized by engineering patient-derived melanoma cells (MC) with a lentiviral vector encoding eGFP under expression control by stemness gene promoter oct4. Their stemness potential was confirmed in vivo by limiting dilution assays. We explored the sensitivity of eGFP+ mCSCs to chemotherapy (CHT), BRAF inhibitor (BRAFi) or CIK cells, as single agents or in sequence, in vitro. First, we treated MCs in vitro with fotemustine or dabrafenib (BRAF-mutated cases); then, surviving MCs, enriched in mCSCs, were challenged with autologous CIK cells. CIK cell activity against chemoresistant mCSCs was confirmed in vivo in two distinct immunodeficient murine models.Results: We visualized eGFP+ mCSCs (14% ± 2.1%) in 11 MCs. The tumorigenic precursor rate in vivo was higher within eGFP+ MCs (1/42) compared with the eGFP− counterpart (1/4,870). In vitro mCSCs were relatively resistant to CHT and BRAFi, but killed by CIK cells (n = 11, 8/11 autologous), with specific lysis ranging from 95% [effector:tumor ratio (E:T), 40:1] to 20% (E:T 1:3). In vivo infusion of autologous CIK cells into mice bearing xenografts from three distinct melanomas demonstrated significant tumor responses involving CHT-spared eGFP+ mCSCs (P = 0.001). Sequential CHT–immunotherapy treatment retained antitumor activity (n = 12, P = 0.001) reducing mCSC rates (P = 0.01).Conclusions: These findings are the first demonstration that immunotherapy with CIK cells is active against autologous mCSCs surviving CHT or BRAFi. An experimental platform for mCSC study and rationale for CIK cells in melanoma clinical study is provided. Clin Cancer Res; 23(9); 2277–88. ©2016 AACR.

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Purpose: The MHC-unrestricted activity of cytokine-induced killer (CIK) cells against chemo-surviving melanoma cancer stem cells (mCSC) was explored, as CSCs are considered responsible for chemoresistance and relapses. Experimental Design: Putative mCSCs were visualized by engineering patient-derived melanoma cells (MC) with a lentiviral vector encoding eGFP under expression control by stemness gene promoter oct4. Their stemness potential was confirmed in vivo by limiting dilution assays. We explored the sensitivity of eGFP+ mCSCs to chemotherapy (CHT), BRAF inhibitor (BRAFi) or CIK cells, as single agents or in sequence, in vitro. First, we treated MCs in vitro with fotemustine or dabrafenib (BRAF-mutated cases); then, surviving MCs, enriched in mCSCs, were challenged with autologous CIK cells. CIK cell activity against chemoresistant mCSCs was confirmed in vivo in two distinct immunodeficient murine models. Results: We visualized eGFP+ mCSCs (14% ± 2.1%) in 11 MCs. The tumorigenic precursor rate in vivo was higher within eGFP+ MCs (1/42) compared with the eGFP− counterpart (1/4,870). In vitro mCSCs were relatively resistant to CHT and BRAFi, but killed by CIK cells (n = 11, 8/11 autologous), with specific lysis ranging from 95% [effector:tumor ratio (E:T), 40:1] to 20% (E:T 1:3). In vivo infusion of autologous CIK cells into mice bearing xenografts from three distinct melanomas demonstrated significant tumor responses involving CHT-spared eGFP+ mCSCs (P = 0.001). Sequential CHT–immunotherapy treatment retained antitumor activity (n = 12, P = 0.001) reducing mCSC rates (P = 0.01). Conclusions: These findings are the first demonstration that immunotherapy with CIK cells is active against autologous mCSCs surviving CHT or BRAFi. An experimental platform for mCSC study and rationale for CIK cells in melanoma clinical study is provided. Clin Cancer Res; 23(9); 2277–88. ©2016 AACR.

Purpose: The goals of this study are i) to analyze the expression of human leukocyte antigen class I (HLA-I) antigen processing machinery (APM) components in surgically removed melanoma tumors, ii) to assess their modulation by Interferons (IFNs) and iii) to determine their value as predictive biomarkers of the clinical response to immunotherapy with checkpoint inhibitors (CI). Methods: The expression levels of HLA class I APM components [HLA-I (ABC) chains, beta(2)microglobulin (β2m), Calnexin, Calreticulin, ERp57, Tapasin, TAP1-2, LMP2, LMP7, LMP10] were evaluated by staining with mAbs and flow cytometry in melanoma cells isolated from surgical biopsies from 16 patients with advanced melanoma. In addition the modulation of these molecules by IFN-γ or IFN-α2 (IFNs) was evaluated. Results: The frequency of cells expressing HLA-I/β2m was >90% in 15/16 Mel tumors. In 1 case extracellular HLA-I/β2m molecules were not detected. The expression levels of extracellular HLA class I APM molecules were quite heterogeneous. Based on Mean Fluorescence Intensity (MFI) of HLA-I/β2m, Mel segregated into 2 groups with high (MFI >200, n=8) and low (MFI Conclusions: We report a wide heterogeneous distribution of HLA-I/β2m expression levels and deficits of APM components in Melanoma, partially restorable by IFNs. Our data support the possibility that defects in HLA expression, not responsive to IFN restoration, may predict low responsiveness to CI. We provide rationale to incorporate the evaluation of HLA-I/β2m and APM in clinical immunotherapy studies with CI. Citation Format: Lidia Giraudo, Loretta Gammaitoni, Bortolot Valentina, Ilenia Iaia, Giulia Cattaneo, Paolo Becco, Susanna Gallo, Alessandro Zaccagna, Alberto Pisacane, Luca Crotto, Soldano Ferrone, Massimo Aglietta, Fabrizio Carnevale Schianca, Dario Sangiolo. Expression and modulation by IFN of HLA class I APM components in melanoma: Potential biomarkers of clinical response to checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 569.

Purpose: We investigate the unknown tumor-killing activity of cytokine-induced killer (CIK) cells against autologous metastatic melanoma and the elusive subset of putative cancer stem cells (mCSC). Experimental Design: We developed a preclinical autologous model using same patient-generated CIK cells and tumor targets to consider the unique biology of each patient/tumor pairing. In primary tumor cell cultures, we visualized and immunophenotypically defined a putative mCSC subset using a novel gene transfer strategy that exploited their exclusive ability to activate the promoter of stemness gene Oct4. Results: The CIK cells from 10 patients with metastatic melanoma were successfully expanded (median, 23-fold; range, 11–117). Primary tumor cell cultures established and characterized from the same patients were used as autologous targets. Patient-derived CIK cells efficiently killed autologous metastatic melanoma [up to 71% specific killing (n = 26)]. CIK cells were active in vivo against autologous melanoma, resulting in delayed tumor growth, increased necrotic areas, and lymphocyte infiltration at tumor sites. The metastatic melanoma cultures presented an average of 11.5% ± 2.5% putative mCSCs, which was assessed by Oct4 promoter activity and stemness marker expression (Oct4, ABCG2, ALDH, MITF). Expression was confirmed on mCSC target molecules recognized by CIK cells (MIC A/B; ULBPs). CIK tumor killing activity against mCSCs was intense (up to 71%, n = 4) and comparable with results reported against differentiated metastatic melanoma cells (P = 0.8). Conclusions: For the first time, the intense killing activity of CIK cells against autologous metastatic melanoma, including mCSCs, has been shown. These findings move clinical investigation of a new immunotherapy for metastatic melanoma, including mCSCs, closer. Clin Cancer Res; 19(16); 4347–58. ©2013 AACR.

Purpose of the study was to explore the activity of cytokine-induced killer (CIK) cells against autologous chemo-surviving melanoma cancer stem cells (mCSCs) in vivo. Elimination of mCSCs is an ambitious goal as considered responsible for chemo-resistance and relapses. CIK cells are ex vivo expanded T-NK lymphocytes with MHC-independent antitumor activity. We provided proof of concept that CIK cells can kill mCSCs in vitro (Gammaitoni et al. Clin Cancer Res 2013) and demonstration of their activity in vivo, within an autologous model, would be relevant in clinical perspective. Experimental procedures and results. In vivo activity of CIK cells against autologous mCSCs was explored in NOD/SCID mice bearing tumors generated by subcutaneous implantation of primary melanoma cells or alternatively surgically resected tumor samples (patient-derived xenografts, PDX). mCSCs were visualized by a lentiviral CSC-detector encoding eGFP under control of stem-gene promoter OCT4 (Gammaitoni et al. Clin Cancer Res 2013). Melanoma cells were engineered with the CSC-detector before implantation or alternatively, in PDX, after removal of residual tumors at the end of experimental treatments. In vivo chemotherapy (CHT) consisted of intravenous (i.v.) Fotemustine (600μg days 1;15). Immunotherapy consisted of i.v. CIK cells (1×107) every 5 days. Antitumor activity was evaluated assessing tumor proliferative index by Ki67 expression in residual tumors. Activity against mCSCs was evaluated assessing the rate of residual GFP+-mCSCs in residual tumors after treatments. In vivo infusion of CIK cells for 2 weeks determined significant tumor response (n = 6, p < 0.0001) that fully involved autologous eGFP+-mCSCs. On the contrary, CHT spared mCSCs that resulted significantly increased in residual tumors compared with mice treated with CIK cells (p = 0.001) or untreated controls (p = 0.001). CIK cells infused after a previous CHT treatment (n = 6) retained significant antitumor activity (p = 0.001) and were capable to revert the CHT-induced enrichment of eGFP+-mCSCs, compared to mice treated with CHT alone (p = 0.01). Similarly, in the PDX model, the infusion (n = 4) of autologous CIK cells exerted significant antitumor activity that involved eGFP+mCSCs (p = 0.009), confirmed by the absence of their increment in residual tumors (p = 0.3). In vitro CHT treatment of melanoma from 8 different patients significantly enriched eGFP+ mCSCs (2.2 ±0.2 fold, p = 0.0018) compared to untreated controls. Melanoma treated with CHT, enriched in eGFP+mCSCs, were efficiently killed by autologous CIK cells with mean killing values ranging from 94±2% (40:1 E/T) to 21%±4 (1:3 E/T). Conclusions. Our findings provide first demonstration that immunotherapy with CIK cells is active against autologous mCSCs, surviving chemotherapy, in vitro and in vivo. We provide an experimental platform to study mCSCs and rationale to design clinical studies with CIK cells against melanoma. Citation Format: Loretta Gammaitoni, Lidia Giraudo, Marco Macagno, Giulia Cattaneo, Valeria Leuci, Francesco Sassi, Alessandro Zaccagna, Alberto Pisacane, Valentina Coha, Fabrizio Carnevale-Schianca, Massimo Aglietta, Dario Sangiolo. Effective immunotherapy with cytokine-induced killer cells against autologous melanoma cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2290.