Dendritoma Vaccine for Cancer: A Hopeful Appraoch Current Cancer Therapy Reviews
Vol. 5, No. 2
mor-cell specific immune-responses that eliminated estab-lished pulmonary metastases.Since then, this research field has expanded quickly anddramatically. This approach has been applied to many tumor models, with encouraging results. These include melanoma[30-35], fibrosarcoma [36, 37], glioma [38, 39], lung carci-noma , colon carcinoma [41-43], hepatocellular carci-noma [44, 45], renal cell carcinoma , and others. Variousmethods have been attempted for the fusion of DCs and tu-mor cells, including chemical [29-31, 36, 38, 39, 41, 42, 45,47-52], physical [32, 33, 43, 46, 49, 53-58], and biological[34, 35] methods. These studies not only generated somefavorable results, but also confirmed the concept that DC-tumor fusion, through which tumor antigens can be presentedto immune systems by both the exogenous and endogenousantigen processing pathways, is a more powerful approach toinduce antitumor immunity than tumor antigen pulsing or tumor-antigen gene transfer [59-65]. Clinical studies usingthe DC-tumor fusion approach have been tested in manydiseases as well, including melanoma [58, 66], malignantglioma [51, 67], breast cancer [68, 69] and renal cell carci-noma [69, 70]. Although some favorable immunologicalresults were observed from these clinical studies, and safetyhas been confirmed to be a non-issue with this approach, theoverall clinical outcomes of these studies have been discour-aging.
ii. Combination of DC-Tumor Fusion with Other Agents
Many approaches have been examined in order to en-hance the antitumor clinical-response induced by DC-tumor fusion vaccines in cancer patients. Cytokines such as granu-locyte-macrophage colony-stimulating factor (GM-CSF),interleukin-2 (IL-2), interleukin-12 (IL-12), or interleukin-18(IL-18) were introduced into the DC-tumor hybrids [30, 36].Cytokines and/or other agents were also used as adjuvants toenhance the activity of DC-tumor fusion. These include IL-2,IL-12, IL-18 [71-75], Toll-like receptor (TLR) agonists suchas CpG oligodeoxynucleotide (ODN) [76, 77], OK432 [78,79] and others.
iii. Unstable Fusion Rate of DC-Tumor Fusion
Although fusion rates as high as 50% by PEG [30, 31,44, 48, 49] and 83.1% by electrofusion  have been re- ported, how to achieve a stable and high DC-tumor cell fu-sion rate still presents a major challenge to researchers, espe-cially when patients’ primary tumor cells are used. With our extensive fusion experience, the highest fusion rate of DCsand primary tumor-cells, either by PEG or by electrofusion,has been below 10% (unpublished data). Due to the lack of an effective way to purify the hybrids from the fusion mix-ture, in most published studies, the vaccines contained verysmall numbers of fused cells, but are contaminated withlarge numbers of un-fused tumor cells, un-fused DCs, self-self fused tumor cells, or self-self fused DCs. The injectionof this mixture could easily overwhelm the immune systemand restrict immune responses from reaching maximum ca- pacity. This argument may represent a good explanation whymost favorable results were achieved in animal models butnot in human clinical trials.
iv. Hybrid Purification from DC-Tumor Fusion
Various methods have been examined to purify the hy- brids from DC-tumor fusions. Some researchers have takenadvantage of the adherent feature of some tumor cell lines byincubating fusion mixture for a short period of time to allowthese cell hybrids to attach to the culture flask, followed byremoval of the non-adherent DCs and collection of theloosely-attached fused cells by pipette aspiration, while leav-ing the tightly attached, unfused tumor cells in the cultureflask [5, 29]. Despite the ease of this method with modelcell-lines, this approach is not practical for clinical settings because most patient primary tumor-cells are non-adherentcells or there is no adherent difference between adherenttumor-cells and fused hybrids. An alternative method to pu-rify DC-tumor hybrids is to fuse DCs and tumor cells fluo-rescently labeled with antibodies specific for DC or tumor markers and sort the unique dual colored hybrids by FACS. This approach is fraught with two problems: 1) labelingof DCs with antibodies may compromise their function; and2) tumor-cell specific markers are not always available, es- pecially for patients’ primary tumor-cells.Another way to purify fused DC-tumor hybrids involvesthe genetic labeling of DCs. Phan
 transfected DCswith a mouse tyrosinase-GFP fusion reporter-gene that isunder the control of a mouse melanoma-specific-tyrosinase promoter and fused the transfected DCs with mouse B16melanoma-tumor cells. As a result, only the hybrid cells ex- pressed GFP, which allowed them to be purified by FACSsorting. Although this approach is very cleverly novel andelegantly marks the hybrids in an extremely accurate man-ner, it is too complicated and time consuming to transfect alarge numbers of patients’ DCs in a clinical setting.
B. DENDRITOMA VACCINEi. PKH Dyes
Our group was the first to report a simple and rapidmethod (dendritoma technology, see below) for purifying thehybrids from a DC-tumor fusion mixture by introducing dis-tinct cyanine PKH fluorescent dyes into the DCs and tumor cells prior to fusion such that the fusion product could bedistinguished from tumor and DC cells allowing for instant purification and isolation of the fused DC-tumor cells. ThePKH dyes developed by Horan [80-82] permit stable, repro-ducible cell-labeling through the incorporation of highlyaliphatic-reporter molecules containing fluorochrome headgroups into the lipid bi-layer of cytoplasmic membranes.Once incorporated into the lipid bilayer, the probes aretrapped within the membrane because of their inherent in-solubility and the probes remain bound to the cell mem- branes the life of the cell.The effects of PKH dye labeling on a variety of mammal-ian cells have been examined. The proliferation of tumor-infiltrating lymphocytes was not affected by labeling withPKH-26GL and no toxic effect was noted in
experi-ments . Cytotoxic effects were not found in labeling leu-kemic cell lines or other cells with PKH dyes [84-86]. A celltracking study in baboons using PKH labeled platelets foundno toxic effect . Oh
 stained several different