Preconditioning Chemotherapy Enhances The Antitumor Activity Of Cytokine-induced Killer Cells Through Modulation Of The Host Immune Microenvironment And The Immunogenicity Of Tumor Cells

Background and ObjectiveIt is now clear that chemotherapy, in addition to its direct cytotoxic effects on tumor cells, could exert a profound influence on antitumor immune response. A number of studies have demonstrated that some chemotherapeutic agents could enhance the antitumor activity of subsequent adoptive cell transfer when used as a preconditioning regimen. The purpose of this study was to investigate the enhancement of the antitumor effect of cytokine-induced killer (CIK) cells induced by preconditioning chemotherapy and to elucidate the underlying mechanisms by which chemotherapeutic agents break down the immunosuppressive barriers to release the full potential of the CIK cell therapy.Materials and Methods1. C57BL/6 mice were inoculated with Lewis cells to establish the murine lung carcinoma model and then randomly divided into six groups. (a) Group Control:treated with normal saline (NS); (b) Group NS-3-CIK:treated with NS followed 3 days by CIK cells; (c) Group NS-7-CIK:treated with NS followed 7 days by CIK cells; (d) Group TP:treated with TP regimen:paclitaxel (PTX) plus cisplatin (DDP); (e) Group TP-3-CIK:preconditioned with TP regimen followed 3 days by CIK cells; (f) Group TP-7-CIK:preconditioned with TP regimen followed 7 days by CIK cells. Tumor size was monitored as indicator of therapeutic response.2. BALB/c wild type and BALB/c nu/nu mice were challenged with CT-26 cells to establish the colon adenocarcinoma model and then randomly divided into four groups. (a) Group NS: administered with NS; (b) Group CIK:administered with CIK cells; (c) Group DDP: administered with DDP; (d) Group DDP-CIK:administered with DDP followed 3 days by CIK cells. Tumor size and weight were used as indicators of therapeutic response.3. C57BL/6 mice were injected with B16 cells to establish the murine melanoma model and then randomly divided into four groups. (a) Group NS:administered with NS; (b) Group CIK: administered with CIK cells; (c) Group DDP:administered with DDP; (d) Group DDP-CIK: administered with DDP followed 3 days by CIK cells. Tumor size was used as indicators of therapeutic response.4. The in vivo trafficking and homing ability of GFP+ CIK cells were observed by fluorescent microscopy.5. Immunohistochemistry was performed to observe the intratumoral infiltration of CD3+ T lymphocytes and FoxP3+ regulatory T (Treg) cells and tumor microvessel density.6. The dynamic changes of endogenous T lymphocytes, dendritic cells (DCs), myeloid-derived suppressor cells (MDSCs) and Treg cells in various tissues after preconditioning chemotherapy were analyzed through flow cytometry.7. The in vitro high, moderate and low toxic concentrations of chemotherapeutic agents were determined by MTT assay and WST-1 assay was used to evaluate the in vitro cytotoxity of CIK cells against drug-treated or untreated tumor cells.8. RT-PCR was employed to detect the expression of immunogenicity-related molecules of tumor cells in mRNA level after treatment with chemotherapeutic agents.Results1. In Lewis lung carcinoma model, CIK cells following TP preconditioning chemotherapy could significantly inhibit the growth of Lewis lung carcinoma (P<0.05), whereas CIK therapy alone or DDP chemotherapy alone failed to induced evident inhibition of tumor growth (P >0.05). At the end of the experiment, the tumor volumes were 3377.82±1603.43mm3 in Group TP-3-CIK,3183.38±806.08mm3 in Group TP-7-CIK,5997.46±1372.90mm3 in Group NS-3-CIK,6206.70±1700.61mm3 in Group NS-7-CIK,6387.09±1019.48mm3 in Group TP and 7087.57±1103.37mm3 in Group NS, respectively.2. In CT-26 colon adenocarcinoma model, CIK cell therapy alone or DDP alone could markedly inhibit the growth of CT-26 colon adenocarcinoma in BALB/c wild type mice (P<0.05), however the combination of DDP preconditioning chemotherapy and CIK cells could induce more significant tumor growth retardation compared with single therapy (P<0.05). At the end of the experiment in BALB/c wild type mice, the tumor volumes were 2115.62±436.61mm3 in Group DDP-CIK,2937.44±773.78mm3 in Group CIK, 2885.26±318.88mm3 in Group DDP and 3867.55±207.56mm3 in Group NS, respectively. In BALB/c nu/nu mice, the combination therapy and both of the single therapies failed to induce significant tumor growth inhibition (P>0.05). At the end of the experiment in BALB/c nu/nu mice, the tumor volumes were 3198.15±863.57mm3 in Group DDP-CIK, 3445.42±786.73mm3 in Group CIK,3298.26±788.13mm3 in Group DDP and 3778.65±819.22mm3 in Group NS, respectively.3. In B16 melanoma model, the combination of DDP preconditioning chemotherapy and CIK cells could significantly inhibit the growth of B16 melanoma (P<0.05), whereas CIK therapy alone or DDP chemotherapy alone failed to induced evident inhibition of tumor growth (P >0.05). At the end of the experiment, the tumor volumes were 2644.41±910.8mm3 in Group DDP-CIK,5634.08±486.89mm3 in Group CIK,5215.31±1118.87mm3 in Group DDP and 5587.62±1390.01mm3 in Group NS, respectively.4. The TP regimen could augment the homing ability of infused CIK cells into tumor and spleen tissues in Lewis lung carcinoma model.5. The TP regimen in Lewis lung carcinoma model could increase the level of T lymphocytes in local tumor tissues, and DDP preconditioning chemotherapy in CT-26 colon adenocarcinoma model could increase the percentages of endogenous T lymphocytes in tumor tissue and tumor-draining lymph nodes (TDLNs). Neither of the precondition exerted influence on tumor microvessel density.6. The DDP pretreatment could up-regulate the percentages of DCs in bone marrows, peripheral bloods and spleen tissues, diminish the levels of MDSCs in bone marrows, peripheral bloods, spleen tissues and TDLNs, and decrease the percentages of Treg cells in tumor and spleen tissues.7. The human lung adenocarcinoma cells displayed an increased sensitivity to the lyses of CIK cells after treatment with chemotherapeutic drugs in vitro.8. Treatment with chemotherapeutic agents could up-regulate the expression of ULBP, Fas, ICAM-1 and DNAM, and down-regulate the expression of Clr-b in mRNA level in human lung adenocarcinoma cells.Conclusions1. Our data indicate that the preconditioning chemotherapy could enhance the antitumor activity of CIK cell therapy in various murine tumor models, and the endogenous T lymphocytes were involved in this enhancement.2. The underlying mechanisms mediating the chemotherapy-induced enhancement of CIK cells' efficacy may involve the augmentation of homing ability of CIK cells to tumor and spleen tissues, and the modulation of percentages of endogenous immune cells in various tissues.3. Treatment of chemotherapeutic agents may sensitize tumor cells to the lyses of CIK cells in vitro through up-regulating in the transcriptional expression of immunogenic molecules of target cells.4. To our knowledge, this is the first study to evaluate the efficacy-enhancing effect of preconditioning chemotherapy on the subsequent adoptive CIK transfer. And this efficacy-enhancing effect may be associated with the modulation of the host immune microenvironment and the immunogenicity of tumor cells induced by chemotherapy.5. This combined chemo-immunotherapy strategy could be easily translated into a clinical setting and is therefore an attractive modality of combined chemo-immunotherapy applicable to patients with malignant cancer.