Host defense peptides for treatment of colorectal carcinoma - a comparative in vitro and in vivo analysis.

Host defense peptides (HDP) constitute effector molecules of the innate immune system. Besides acting against microbia and fungi, they exhibit broad and selective oncolytic activity. The underlying mechanism is at least partially attributable to elevated surface-exposed levels of phosphatidylserine (PS) on tumor targets. In this study, comprehensive analysis of NK-2-based derivatives (C7A, C7A-D21K, and C7A-Δ) was done on patient-derived ultra-low passage colorectal carcinoma (CRC) cell lines. Peptides were designed to improve antitumoral potential. Mellitin was used as positive control and a non-toxic peptide (NK11) served as negative control. Subsequently, effectiveness of local HDP application was determined in xenopatients. Generally, CRC lines displayed a heterogeneous pattern of surface-exposed PS, which was usually below standard CRC cells. Of note, five out of seven cell lines were susceptible towards HDP-mediated lysis (lytic activity of peptides: C7A-D21K > C7A-Δ= C7A). Oncolytic activity correlated mostly with surface-exposed PS levels. Apoptosis as well as necrosis were involved in killing. In an in vivo experiment, substantial growth inhibition of HROC24 xenografts was observed after HDP therapy and, surprisingly, also after NK11 treatment. These promising data underline the high potential of HDPs for oncolytic therapies and may provide a rationale for optimizing preclinical treatment schedules based on NK-2.

Combinations of TLR ligands: a promising approach in cancer immunotherapy.

Toll-like receptors (TLRs), a family of pattern recognition receptors recognizing molecules expressed by pathogens, are typically expressed by immune cells. However, several recent studies revealed functional TLR expression also on tumor cells. Their expression is a two-sided coin for tumor cells. Not only tumor-promoting effects of TLR ligands are described but also direct oncopathic and immunostimulatory effects. To clarify TLRs' role in colorectal cancer (CRC), we tested the impact of the TLR ligands LPS, Poly I:C, R848, and Taxol on primary human CRC cell lines (HROC40, HROC60, and HROC69) in vitro and in vivo (CT26). Taxol, not only a potent tumor-apoptosis-inducing, but also TLR4-activating chemotherapeutic compound, inhibited growth and viability of all cell lines, whereas the remaining TLR ligands had only marginal effects (R848 > LPS > Poly I:C). Combinations of the substances here did not improve the results, whereas antitumoral effects were dramatically boosted when human lymphocytes were added. Here, combining the TLR ligands often diminished antitumoral effects. In vivo, best tumor growth control was achieved by the combination of Taxol and R848. However, when combined with LPS, Taxol accelerated tumor growth. These data generally prove the potential of TLR ligands to control tumor growth and activate immune cells, but they also demonstrate the importance of choosing the right combinations.

Combining bacterial-immunotherapy with therapeutic antibodies: a novel therapeutic concept.

Immunotherapeutic strategies become more and more important for cancer treatment. Therapeutic monoclonal antibodies (mAbs) like Panitumumab binding and blocking the EGF-receptor are in routine clinical use for the treatment of colorectal carcinoma (CRC). Also, bacterial therapy proved beneficial for experimental treatment of different tumor entities. The latter has been attributed to an activation of the immune system. Here, we describe a combination of both immunotherapeutic approaches in order to develop a novel targeted therapy for CRC. The therapeutic mAbs Trastuzumab and Panitumumab were conjugated to heat-inactivated bacteria expressing protein A or protein G. The potential of the conjugates was tested in comparison to the single components both in vitro and in vivo using a panel of patient-derived CRC cell lines. Antitumoral effects observed in vitro were strictly dependent on the presence of bacteria. Generally, effects could be enhanced by the addition of human lymphocytes. Detailed analysis of effector cells in autologous and allogeneic long-term stimulated lymphocyte cultures revealed the predominance of NK-cell-like cytolytic effectors. Reactivity was observed both against CRC target cells but also against the NK cell target K562. Similarly, in a subsequent in vivo study we observed substantial tumor growth delay accompanied by an increase in circulating NK cells. Contrary to this, the monotherapy with mAb alone caused only marginal effects and the treatment with bacteria was comparable to the mock-treated control. These data demonstrate successful targeting of CRC by bacteria/mAb conjugates. This novel concept may be interesting for future clinical approaches. Additionally, it illustrates the effectiveness of NK cells for cancer immunotherapy.

Bacterial immunotherapy of gastrointestinal tumors.

BACKGROUND: Cancer immunotherapy using bacteria dates back over 150 years. The deeper understanding on how the immune system interferes with the tumor microenvironment has led to the re-emergence of bacteria or their related products in immunotherapeutic concepts. In this review, we discuss recent approaches on experimental bacteriolytic therapy, emphasizing the specific interplay between bacteria, immune cells and tumor cells to break the tumor-induced tolerance. RESULTS: Experimental research during the last decades demonstrated beneficial but also adverse influence of bacteria on tumor growth. There is a strong correlation between chronic infections and tumor incidence. However, acute bacterial infections have favourable effects on tumor growth often contributing to complete remission. Tumor regression is usually attributable to both direct tumor cell killing (via apoptosis and/or necrosis, depending on the applied bacteria) and indirect immune stimulation. This includes (I) elimination of immunosuppressive immune cells (i.e. tumor-associated macrophages, myeloid-derived suppressor, and regulatory T cells), (II) suppression of Th2-directed cytokine secretion (TGFα, IL10), (III) providing a pro-inflammatory micro-milieu (tumor infiltrating neutrophils) and (IV) supporting the influx of cytotoxic T cells into tumors. This finally forces the development of an immunological memory and may provide long-term protection against cancer. CONCLUSION: Immunotherapy using bacteria is still a double-edged sword. Experiences from the last years have substantially contributed to when bacteria and defined components thereof might be integrated into immunotherapeutic concepts. Attempts in transferring this approach into the clinics are on their way.

Semiallogenic fusions of MSI(+) tumor cells and activated B cells induce MSI-specific T cell responses.

BACKGROUND: Various strategies have been developed to transfer tumor-specific antigens into antigen presenting cells in order to induce cytotoxic T cell responses against tumor cells. One approach uses cellular vaccines based on fusions of autologous antigen presenting cells and allogeneic tumor cells. The fusion cells combine antigenicity of the tumor cell with optimal immunostimulatory capacity of the antigen presenting cells.Microsatellite instability caused by mutational inactivation of DNA mismatch repair genes results in translational frameshifts when affecting coding regions. It has been shown by us and others that these mutant proteins lead to the presentation of immunogenic frameshift peptides that are - in principle - recognized by a multiplicity of effector T cells. METHODS: We chose microsatellite instability-induced frameshift antigens as ideal to test for induction of tumor specific T cell responses by semiallogenic fusions of microsatellite instable carcinoma cells with CD40-activated B cells. Two fusion clones of HCT116 with activated B cells were selected for stimulation of T cells autologous to the B cell fusion partner. Outgrowing T cells were phenotyped and tested in functional assays. RESULTS: The fusion clones expressed frameshift antigens as well as high amounts of MHC and costimulatory molecules. Autologous T cells stimulated with these fusions were predominantly CD4(+), activated, and reacted specifically against the fusion clones and also against the tumor cell fusion partner. Interestingly, a response toward 6 frameshift-derived peptides (of 14 tested) could be observed. CONCLUSION: Cellular fusions of MSI(+) carcinoma cells and activated B cells combine the antigen-presenting capacity of the B cell with the antigenic repertoire of the carcinoma cell. They present frameshift-derived peptides and can induce specific and fully functional T cells recognizing not only fusion cells but also the carcinoma cells. These hybrid cells may have great potential for cellular immunotherapy and this approach should be further analyzed in preclinical as well as clinical trials. Moreover, this is the first report on the induction of frameshift-specific T cell responses without the use of synthetic peptides.

Avitalized bacteria mediate tumor growth control via activation of innate immunity.

Acute bacterial infections have beneficial effects on tumor patients. To eliminate side effects evoked by viable microbes, we here assessed the immunotherapeutic potential of inactivated bacteria on colorectal carcinomas. Our In vitro results indicate a cell-specific direct cytotoxicity towards tumor cells presented by G1-arrest. Antitumoral activity was boosted in the presence of leukocytes. Long time stimulations revealed massive activation of NK cells even in complete autologous settings. In vivo, repetitive local treatment mediated tumor growth control. Evaluation of residual tumors identified increased infiltrates, with NK cells (CD49b(+), NKG2D(+)) being the main responding cell population. Substantial NK cell-mediated delay of tumor growth was also achieved in T-cell deficient mice xenografted with human colorectal carcinomas. Of note, local as well as systemic therapy mediated tumor growth control. These data highlight the potential of avitalized bacteria to especially activate the immune system's innate arm and they should be considered for future integrated immunotherapy.

Bacteriolytic therapy of experimental pancreatic carcinoma.

AIM: To investigate the effectiveness of Clostridium novyi (C. novyi)-NT spores for the treatment of established subcutaneous pancreatic tumor in the syngeneic, immunocompetent Panc02/C57Bl/6 model. METHODS: C. novyi-NT spores were applied intravenously to animals carrying established pancreatic tumors of three different sizes. Systemic immune responses in peripheral blood and spleen were examined by flow cytometry. Supplementary, cytotoxic activity of lymphocytes against syngeneic tumor targets was analyzed. RESULTS: Application of spores identified, that (1) small tumors (< 150 mm(3)) were completely unaffected (n = 10); (2) very large tumors (> 450 mm(3)) responded with substantial necrosis followed by shrinkage and significant lethality most likely due to tumor lysis syndrome (n = 6); and (3) an optimal treatment window exists for tumors of approximately 250 mm(3) (n = 21). In this latter group, all tumor-bearing animals had complete tumor regression and remained free of tumor recurrence. In subsequent tumor rechallenge experiments a significant delay in tumor growth compared to the initial tumor cell inoculation was observed (tumor volume at day 28: 197.8 +/- 87.3 mm(3) vs 500.1 +/- 50.9 mm(3), P < 0.05). These effects were accompanied by systemic activation of immune response mechanisms predominantly mediated by the innate arm of the immune system. CONCLUSION: The observed complete tumor regression is encouraging and shows that immunotherapy with C. novyi-NT is an interesting strategy for the treatment of pancreatic carcinomas of defined sizes.

Cryopreservation of human colorectal carcinomas prior to xenografting.

BACKGROUND: Molecular heterogeneity of colorectal carcinoma (CRC) is well recognized, forming the rationale for molecular tests required before administration of some of the novel targeted therapies that now are rapidly entering the clinics. For clinical research at least, but possibly even for future individualized tumor treatment on a routine basis, propagation of patients' CRC tissue may be highly desirable for detailed molecular, biochemical or functional analyses. However, complex logistics requiring close liaison between surgery, pathology, laboratory researchers and animal care facilities are a major drawback in this. We here describe and evaluate a very simple cryopreservation procedure for colorectal carcinoma tissue prior to xenografting that will considerably reduce this logistic complexity. METHODS: Fourty-eight CRC collected ad hoc were xenografted subcutaneously into immunodeficient mice either fresh from surgery (N = 23) or after cryopreservation (N = 31; up to 643 days). RESULTS: Take rates after cryopreservation were satisfactory (71%) though somewhat lower than with tumor tissues fresh from surgery (74%), but this difference was not statistically significant. Re-transplantation of cryopreserved established xenografts (N = 11) was always successful. Of note, in this series, all of the major molecular types of CRC were xenografted successfully, even after cryopreservation. CONCLUSIONS: Our procedure facilitates collection, long-time storage and propagation of clinical CRC specimens (even from different centres) for (pre)clinical studies of novel therapies or for basic research.

Generation of highly pure fusions of colorectal carcinoma and antigen-presenting cells.

PURPOSE: The induction of potent T cell responses against tumors is the goal of tumor immunotherapy. One approach is the fusion of antigen-presenting cells (APCs) with tumor cells. Hybrid cells combine the antigenicity of tumors with the immunostimulatory capacity of APCs. However, contaminating unfused cells present in the fusion reaction may prevent the induction of antitumoral immune responses. Here, we present a simple and effective protocol to substantially elevate the purity of hybrid cells. METHODS: Colorectal tumor cell lines and CD40-activated B cells as APCs were fused using polyethylene glycol. Important parameters including cell numbers, concentrations, and handling and detection procedures were optimized. Combination of these optimized fusion conditions with both magnetic cell sorting and selective adherence delivered very pure preparations of APC/tumor cell hybrids. The T cell stimulatory capacity of these hybrids was tested using ELISpot. RESULTS: The optimization of the fusion resulted in maximal fusion efficiencies of 31.6% (n = 10, range 13.5-46.6%). Prelabeling of APCs with magnetic beads allowed for easy elimination of up to 94.3% of unfused tumor cells from the cell mixture by magnetic separation. Hybrid cell capacity to firmly adhere to plastic was then used to remove unfused B cells from the remaining cell mixture by simple washing. The obtained 85.0% pure hybrids cells readily induced antitumoral T cell responses. CONCLUSIONS: Our protocol delivers pure hybrid cell preparations with strong immunostimulatory potential. In subsequent experiments, the ability of hybrid cells to stimulate specific antitumoral T cell responses must be tested in vivo.

Histone deacetylase inhibitors sensitize tumour cells for cytotoxic effects of natural killer cells.

Histone deacetylase inhibitors (HDIs) are emerging as potent anti-tumour agents which induce cell cycle arrest, differentiation and/or apoptosis in many tumour cells. Furthermore, they render tumour cells more sensitive to other therapeutic regimens like ionizing radiation, chemotherapy and recombinant tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Here, we show that the HDIs suberoylanilide hydroxamic acid (SAHA; vorinostat), sodium butyrate (NaB) and MS-275 sensitized DAOY and PC3 tumour cells for the cytotoxic effects of IL-2-activated PBMCs. In (51)Cr-release assays, blockade of the activating NK receptors DNAM-1, NKG2D and the NCRs completely abrogated tumour cell lysis, revealing that NK cells were the main effector cells involved. HDIs increased the tumour surface expression of ligands for the activating NK receptors NKG2D and DNAM-1 thereby facilitating tumour cell recognition by NK cells. These results suggest that the combination of HDIs and immunotherapy may be an effective strategy for anti-cancer therapy.

Histone deacetylase inhibitors induce cell death and enhance the apoptosis-inducing activity of TRAIL in Ewing's sarcoma cells.

PURPOSE: The present in vitro study was conducted to evaluate the effects of the histone deacetylase inhibitors (HDIs) suberoyl anilide hydroxamic acid (SAHA), sodium butyrate (NaB) and MS-275 applied as single agents or in combination with TRAIL in Ewing's sarcoma. METHODS: Cytotoxic activities were assessed by cytofluorometric analysis of propidium iodide uptake, DNA fragmentation and mitochondrial depolarisation as well as by measuring caspase-9 and -3 activities. Cell-surface expression of TRAIL receptors was determined by cytofluorometry, and histone H4 acetylation was assessed by western blot. RESULTS: All three HDIs potently induced cell death in the two cell lines explored, SK-ES-1 and WE-68. However, they seemed to differ in their modes of action. SAHA and NaB induced mitochondrial depolarisation as well as caspase-9 and -3 activities, and their cytotoxic effects could be significantly reduced by the pan-caspase inhibitor z-VAD-fmk. MS-275 was a much weaker inducer of caspase-9 and -3 activities as well as mitochondrial injury; consistently, z-VAD-fmk had little effect on MS-275-mediated activities. Combined treatment of HDIs and TRAIL led to an additive effect in SK-ES-1 cells and a supra-additive effect in WE-68 cells. Yet, HDIs did not increase cell-surface expression of TRAIL receptor 2, but rather decreased it. Selective inhibition of caspase-8 in WE-68 cells and HDI treatment of CADO-ES-1 cells, a Ewing's sarcoma cell line deficient in caspase-8 expression, revealed that caspase-8 was not required for HDI-mediated apoptosis. CONCLUSIONS: These results suggest that HDIs may be considered as a novel treatment strategy for Ewing's sarcoma either applied as monotherapy or in combination with TRAIL.