Tumor cell lysate-pulsed dendritic cells induce a T cell response against colon cancer in vitro and in vivo

Abstract

To investigate whether tumor cell lysate-pulsed (TP) dendritic cells (DCs) induce cytotoxic T lymphocyte (CTL) activity against colon cancer in vitro and in vivo. Hematopoietic progenitor cells were magnetically isolated from BALB/c mice bone marrow cells. These cells were cultured with cytokines GM-CSF, IL-4, and TNFalpha to induce their maturation. They were analyzed by morphological observation and phenotype analysis. DCs were pulsed with tumor cell lysate obtained by rapid freezing and thawing at a 1:3 DC:tumor cell ratio. CTL activity and interferon gamma (IFNgamma) secretion was evaluated ex vivo. In order to determine whether or not vaccination with CT26 TP DCs induce the therapeutic potential in the established colon tumor model, CT26 colon tumor cells were implanted subcutaneously (s.c.) in the midflank of naïve BALB/c mice. Tumor-bearing mice were injected with vaccination with CT26 TP DCs on days 3 and 10. Tumor growth was assessed every 2-3 days. Finally, CTL activity and IFNgamma secretion were evaluated in immunized mice. Hematopoietic progenitor cells from mice bone marrow cells cultured with cytokines for 8 days showed the character of typical mature DCs. Morphologically, these cells were large with oval or irregularly shaped nuclei and with many small dendrites. Phenotypically, FACS analysis showed that they expressed high levels of MHC II, CD11b, CD80, and CD86 antigen, and were negative for CD8alpha. However, immature DCs cultured with cytokines for 5 days did not have typical DCs phenotypic markers. Ex vivo primed T cells with CT26 TP DCs were able to induce effective CTL activity against CT26 tumor cells, but not B16 tumor cells (E:T = 100:1, 60.36 +/- 7.11% specific lysis in CT26 group vs. 17.36 +/- 4.10% specific lysis in B16 group), and produced higher levels of IFNgamma when stimulated with CT26 tumor cells but not when stimulated with B16 tumor cells (1210.33 +/- 72.15 pg/ml in CT26 group vs. 182.25 +/- 25.51 pg/ml in B16 group, P < 0.01). Vaccination with CT26 TP DCs could induce anti-tumor immunity against CT26 colon tumor in murine therapeutic models (tumor volume on day 19: CT26 TP DCs 342 +/- 55 mm(3) vs. the other control groups, P < 0.05). In addition, all splenic CD3(+) T cells obtained from mice vaccinated with CT26 TP DCs produced high levels of IFNgamma and shown specific cytotoxic activity against CT26 tumor cells, but no cytotoxic activity when stimulated with B16 tumor cells. Tumor cell lysate-pulsed DCs can induce tumor-specific CTL activity against colon cancer in vitro and in vivo.

&lt;i&gt;In vitro&lt;/i&gt; analysis of T cell responses induced by breast tumor cell lysate pulsed with autologous dendritic cells

“…DC yield was 5% -6% of initial culture of PBMCs which was comparable to results obtained by others [28].…”

“…In the last few years evidence that many murine and human tumors may express immunogenic tumor antigens that can be used as targets for tumor specific T lymphocytes has led to the development of different vaccination strategies for antitumor therapy. Because tumor specific antigens is frequently not available, most immunization approaches including this in vitro study have used DCs charged in vitro with whole tumor lysate [16,28,30,31]. It should be noted that we performed our experiments under autologous condition using an experimental in vitro study in which DCs, T cells and breast tumor and normal cells were derived from same individual.…”

“…In all 5 patients with breast cancer, they failed to kill autologous normal cells (specific lysis = 0.6% -8.3%) and exhibited variable amounts of cytotoxic activity against NK sensitive K562 cell line (specific lysis = 6.5% -21%). Previous studies reported that tumor antigen pulsed DC primed T cells exhibited variable amounts of cytotoxicity in murine model [28] as well as in different cancer patients for example 20% -60% in pancreas [34], 40% in multiple myeloma [35], 33% in melanoma and 41% -58% in breast [30,33] cancers. It should be noted that in these studies specific lysis was determined by 51 Cr-release assay which gives somehow lower cytotoxic activity values than flow cytometry.…”

Immunotherapy with tumor cell lysate-pulsed CD8α+ dendritic cells modulates intra-tumor and spleen lymphocyte subpopulations

“…DCs are the most potent APCs mediating the effective in vitro and in vivo immune responses [20,21]. These cells are used for antigen-specific immunotherapy of cancer because they are the most efficient APCs that can induce primary immune responses [22,23].…”

Cytokine-induced killer cells co-cultured with complete tumor antigen-loaded dendritic cells, have enhanced selective cytotoxicity on carboplatin-resistant retinoblastoma cells

“…In addition, DCs can be loaded with antigens (DC-Ag) that may increase the DC's specificity. This in turn enhances the targeting of killing cancer cells (14)(15)(16). Functional T-lymphocytes can be gener-…”