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Conventional cytotoxic drugs preferentially eliminate differentiated cancer cells but spare relatively more resistant stem-like cancer cells capable to initiate recurrence. Due to cancer cell plasticity, the stem-like phenotype can be also acquired by cancer cells refractory to treatment with cytotoxic drugs. We investigated whether drugs inhibiting receptor tyrosine kinases could be used to target human colon cancer cells initiating cancer regrowth following conventional cytotoxic treatment. The moderately differentiated cell line HT-29 and poorly differentiated cell line HCT-116 were exposed to 5-fluorouracil (5-FU). Cells that resisted the exposure to 5-FU were subsequently treated with imatinib or sunitinib. Both drugs reduced clonogenicity of 5-FU-refractory cells under normoxic and hypoxic culture conditions. The expression of numerous stemness-related genes was upregulated in cancer cells following the exposure to 5-FU, and remained at a high level in 5-FU-refractory cells undergoing renewal under normoxia, but decreased spontaneously under hypoxia. Imatinib downregulated the expression of stemness-related genes in cells undergoing renewal under normoxia. A combination of imatinib with PRI-2191, an analogue of 1,25-dihydroxyvitamin D3, downregulated stemness-related genes in HCT-116/5-FU cells more efficiently than imatinib alone. A synthetic analogue of 1,25-dihydroxyvitamin D2 (PRI-1906) abolished the effect of imatinib on gene expression in HCT-116/5-FU cells undergoing renewal under normoxia. Sunitinib promoted shift of phenotype of HT-29/5-FU cells undergoing renewal toward stem-like one. It suggests that the phenotype shift toward stemness induced by sequential sunitinib treatment following 5-FU treatment could increase a risk of cancer recurrence. In contrast to sunitinib, imatinib could be used both to interfere with cancer regrowth after conventional chemotherapy and to downregulate the expression of stemness-related genes in residual colon cancer cells capable to initiate cancer recurrence. The findings suggest that imatinib could also be combined with vitamin D analogue PRI-2191 to prevent recurrence more efficiently than imatinib alone and to compensate for vitamin D deficiency resulting from imatinib treatment.

We have previously found that ex vivo expanded human CD4+CD25+Treg cells suppress proliferation of lymphoma B-cell lines. Here we demonstrate that the immunomodulatory drug lenalidomide potentiates suppression of lymphoma B-cell proliferation by freshly isolated CD4+CD25+Tregs, as well as suppression by Tregs expanded polyclonally in the presence of rapamycin from CD4+CD25+T cells or CD4+CD25+CD127loT cells. The regulation of lymphoma cell proliferation by Tregs pre-expanded with "third-party" allogeneic MoDCs in the presence of rapamycin was also potentiated by lenalidomide. Lenalidomide contributed to the suppression exerted by Tregs despite concomitant downregulation of Treg proliferation. Lenalidomide did not reduce the suppression of conventional T cells by expanded Tregs. The exposure of polyclonally expanded Tregs to lenalidomide did not significantly alter their phenotype. There was no uniform pattern of lenalidomide effect on Treg-mediated regulation of lymphoma B cells freshly isolated from patients. Freshly isolated lymphoma cells activated with multimeric CD40L and IL-4 to support their survival in vitro varied in their sensitivity to lenalidomide, and the regulatory effect of Tregs on such lymphoma cells ranged from suppression to help in individual patients. Lenalidomide potentiated or attenuated Treg effects on the survival of freshly isolated lymphoma cells. A combination of lenalidomide treatment with adoptive transfer of CD4+CD25+Tregs or CD4+CD25+CD127loTregs expanded ex vivo could be used to suppress proliferation of residual lymphoma in select patients with lymphoma responsive to the regulation by Tregs and sensitive to lenalidomide.

A b s t r a c t Opiate analgesics like morphine or fentanyl are the most widely used medicines for relieving severe acute or chronic pain, including cancer pain. Unfortunately, chronic pain treatment is associated with fast development of tolerance that creates the need to escalate the treatment doses. In addition, opiates may stimulate progression of cancer. There - fore, a new type of effective analgesic especially designed for chronic cancer pain treatment is needed. In this paper, a new opioid peptide analogue has been described as a new analgesic. The compound is characterized by very high agonist affinities to MOR and also high, but ten times lower affinity to DOR. Affinity to hNK1 as an antagonist is on the level of C-terminal hexapeptide fragment analogue of Substance P. The compound expressed reasonable antipro - liferative properties toward various cancer cells. Interestingly, the peptide did not interfere with the proliferation of fibro - blasts. Therefore, the compound should be considered as a new analgesic for treatment of cancer-related pains with adjuvant anticancer properties which may support cancer treatments.

Dendritic cells may be successfully used to induce in vivo-specific anti-tumor responses when combined with the appropriate antigen in the appropriate context. The purpose of this study was to evaluate efficacy of peptide-loaded DC vaccine in high-risk stage III melanoma patients after lymph node dissection (LND). HLA-A2+, -A1+, or -A3+ melanoma patients (N=22), stage III, N1b-N3, received 5–16 (median: 11) DC vaccines loaded with MHC class-I-restricted melanoma peptides respective to the patient’s haplotype, and with autologous tumor lysate, if available. Vaccinated patients were matched to unvaccinated stage III controls (22 of 869) by sex, number of metastatic lymph nodes, extracapsular involvement, LND type, Breslow stage, and ulceration. Vaccination elicited cutaneous delayed-type hypersensitivity (DTH) or/and IFN-γ-producing CD8+ cell response to melanoma peptides in 15 of 22 patients. Three-year overall survival (OS) rate was 68.2% in the vaccinated group versus 25.7% in the control group, P value accounting for matching: 0.0290. In a Cox regression model, hazard ratio (HR) for death of vaccinated patients was 0.31 [95% confidence interval (CI): 0.10–0.94]. The corresponding values for 3-year disease-free survival rate were 40.9 versus 14.5%, P=0.1083; HR of recurrence for vaccinated, 0.46 (95% CI: 0.18–1.22). There was no grade>1 toxicity. The DC/peptide vaccine was well tolerated and elicited immune responses to melanoma antigens. Vaccinated patients had significantly longer OS after LND than the matched controls, but a significant improvement in the primary endpoint DFS was not achieved.

Conventional anti-cancer drugs preferentially eliminate differentiated cancer cells but those cells that are spared (i.e. cancer stem cells: CSC), initiate recurrence. We tested whether drugs that target receptor tyrosine kinases (RTKs) involved in developmental signaling cascades and activated in CSC, could be used to silence and/or to eliminate colorectal cancer cells refractory to conventional treatment with cytoreductive drugs. A sequential treatment model was thereby developed with doxorubicin (DOX) and imatinib. CT-26 mouse colon carcinoma cells were pre-treated with DOX to select DOX-refractory cells with CSC properties, which were then subsequently treated with RTK inhibitor imatinib, where their regrowth was found to be inhibited. Under both normoxic and hypoxic conditions, imatinib potently inhibited clonogenicity of DOX-refractory CT-26 cells. Treatment with DOX did not eliminate tumorigenic CT-26 cells, since CT-26 cells pre-exposed to DOX in vitro, when inoculated subcutaneously, induced tumors in 90 % of mice, as opposed to a 100 % rate in the case of chemonaive CT-26 cells. In mice inoculated with chemonaive CT-26 cells, tumor formation was not prevented by imatinib. However, imatinib prevented tumor formation in 50 % of mice inoculated with CT-26 cells pre-exposed to DOX in vitro, with the remaining 50 % mice showing delayed tumor formation. These results suggest that the sequential use of the drug imatinib, as a drug targeting cancer cells expressing stem cell features after conventional cytoreductive treatment, is a promising future strategy for preventing tumor recurrence.

Marker genes, commonly used to detect circulating tumour cells in RT-PCR-based tests: squamous-cell carcinoma antigen, epidermal growth factor receptor, mammaglobin, small breast epithelial mucin, but not carbonic anhydrase 9, were shown to be expressed in normal, mitogen-stimulated peripheral blood mononuclear cells (PBMNC). Thus, considering the inflammatory reactions often accompanying cancer development, to reduce false-positive results of the metastatic tumour cell tests, molecular markers should be validated not against normal peripheral blood, but against activated lymphoid cells, such as in vitro mitogen-stimulated PBMNC.

Dendritic cell (DC) counts and function were assayed in peripheral blood of lymphoma and solid tumor patients before and after chemotherapy. The DC counts declined significantly within the first week from the start of chemotherapy, recovered in the second week, and exceeded the baseline values in the third week. DC recovery was usually similar after the first and after the last cycle of chemotherapy. DC1 and DC2 subsets followed the pattern of reconstitution found for the DC population as a whole. Monocytes and granulocytes recovered 1-2 weeks later than DC. The primary proliferative response to keyhole lympet hemocyanin (KLH), totally DC-dependent, declined within the first week from the start of chemotherapy, and in the majority of patients (including those initially unresponsive) recovered along with DC counts. The recovered responsiveness to KLH, but not to anti-CD3 antibody, disappeared at the end of chemotherapy in lymphoma and some solid tumor patients. Prolonged depletion of CD4+ T cells could contribute to the loss of responsiveness in lymphoma patients receiving multiple cycles of chemotherapy. However, in some solid tumor patients, the reactivity to KLH was absent, despite the reconstitution of both DC and CD4+ T-cell counts. Our data show that numerical reconstitution of DC is not necessarily accompanied by functional recovery. The early recovery of DC should be considered while designing protocols for DC collection for immunotherapy.

It has been previously shown that GM-CSF promotes differentiation and survival of human peripheral blood dendritic cells (DC) in vitro1. Human bone marrow progenitors of dendritic cells were identified in semi-solid cultures supplemented with conditioned media from PHA-stimulated leukocytes2. GM-CSF and TNF-α have been shown to cooperate in the regulation of DC growth from CD34+ progenitors in vitro3,4,5.

A feasibility of hybrid cell vaccination performed in remission following a standard chemotherapy (CHT) is currently being studied in patients with indolent B cell lymphomas at our institution under approval of the Ethics Committee. Adequate samples of fresh tumor tissue were collected from 16 patients. Seven patients were vaccinated with irradiated lymphoma/dendritic cell (DC) hybrids, and whenever the amount of collected viable lymphoma cells was not sufficient to complete vaccination with the use of cell hybrids, booster injections were alternatively performed with the use autologous DCs pulsed with tumor lysate. Autologous lymphoma cells were electrofused with autologous and/or allogeneic DCs, depending on accessibility of autologous DCs. Monocyte-derived DCs were basically generated from the peripheral blood adherent cells stimulated with GM-CSF and IL-4, and if available, from the bone marrow (BM) adherent cells. If only possible, autologous DCs were also generated from the BM non-adherent cells or from a sample of leukapheresis product collected for transplantation cultured with GM-CSF, TNF-α, SCF and FLT3-L to induce differentiation of DC progenitors. DC hybrids and/or DCs preincubated with tumor lysate were administered to uninvolved lymph nodes under USG guidance. Four patients failed to achieve a complete remission after initial therapy and the vaccination was discontinued due to disease progression (after 2, 2, 6, and 5 cycles). Three patients were vaccinated during remission following CHT. At present - 16, 18, and 10 months after starting the immunization, and after 8, 9, and after 7 cycles of vaccination, one patient with mantle cell lymphoma (MCL) is symptomless with persistent bone marrow (BM) involvement, one patient with MCL presenting with IgM paraproteinemia is disease-free with residual BM involvement (Fig 1), and one patient with follicular lymphoma is disease-free. Cutaneous delayed type hypersensitivity (DTH) response to DC/lymphoma hybrids exceeded the response to DCs alone in 3 of 7 vaccinated patients, and interferon-γ producing CD8+ cells specific to autologous lymphoma cells were demonstrated in one patient. In another patient, cutaneous DTH reaction was accompanied by transient pruritus and swelling of the injected lymph node two days after the sixth booster injection. No other adverse reactions to vaccinations were observed. Vaccination using the DC/lymphoma hybrids appears feasible, safe, and capable of inducing lymphoma cell specific immune responses. Further evaluation of efficacy in terms of disease control is mandatory. Fig 1. Disease and treatment course of a representative case Fig 1. Disease and treatment course of a representative case