Background Strong evidence supports the DC-tumor fusion cross vaccination strategy, but the best fusion product components to utilize remains controversial. and the additional parts were compared. Results Except for DC-tumor hybrids, the non-adherent cell portion comprising primarily unfused DCs also contributed a lot in antitumor immunity. Purified hybrids supplemented hWNT5A with the non-adherent cell populace elicited the most powerful antitumor immune response. After irradiation and electro-fusion, tumor cells underwent necrosis, and the unfused DCs phagocytosed the necrotic tumor cells or tumor debris, which resulted in significant DC maturation. This may be the immunogenicity mechanism of the non-adherent unfused DCs portion. Conclusions The non-adherent cell portion (containing primarily unfused DCs) from total DC/tumor fusion products had enhanced immunogenicity that resulted from apoptotic/necrotic tumor cell phagocytosis and improved DC maturation. Purified fusion hybrids supplemented with the non-adherent cell populace enhanced the antitumor immune responses, avoiding unneeded use of the tumor cell portion, which has many drawbacks. Purified hybrids supplemented with the non-adherent cell portion may represent a better approach to the DC-tumor fusion cross vaccination strategy. Intro Dendritic cell (DC)-tumor fusion hybrids have shown advantages among DC-based tumor vaccination strategies. Using the fusion approach, multiple Tumor connected antigens (TAAs), including those yet unidentified, are endogenously processed by major histocompatibility complex (MHC) I and II pathways in the context of co-stimulatory molecules [1], [2], [3]. Several animal studies and early medical tests have shown motivating results from DC and tumor cell fusion [4], [5] [6], [7], [8], [9], [10], [11], [12], [13], [14], [15]. According to earlier studies, the fusion effectiveness (including electro-fusion and chemical fusion) between DC and tumor cells is definitely relatively low, at less than 50% [2], [16], so the total DC-tumor fusion products consist of DC-tumor fusion hybrids, unfused DCs and tumor cells, and DC-DC or tumor-tumor self-fusion, as well as debris and lysate from cells that pass away during the process. However, the degree to which the hybrids themselves along with other parts are responsible for inducing anti-tumor immunity is not well understood. In addition, identification of the best parts that should be used is controversial, and various fractions from the total fusion products, including purified cross cells [8], [9], [16], [17], [18], the adherent cell portion [2], [19], [20] or the entire fusion combination [7], [21], [22], [23], have been used in earlier studies. To the best of our knowledge, any attempt at fusion requires DCs and tumor cells to be mixed together, so potential Lersivirine (UK-453061) co-stimulation and antigen demonstration is possible even though no fusion happens. Thus, it is difficult to know whether reported restorative responses Lersivirine (UK-453061) result from the presence of a fused DC-tumor component or from unfused DCs showing antigen through uptake of tumor-associated material or additional parts in the fusion combination. In order to investigate the functions of hybrids themselves along with other fusion product parts in anti-tumor immunity and to determine which parts should be used in the DCs-tumor fusion vaccination, patient-derived DCs and auto breast tumor cells were electro-fused to generate the fusion hybrids and then fluorescence triggered cell sorting FACS was used to purify the truely fused cells. We then compared the antitumor immune reactions induced by purified hybrids to that of additional parts in the total fusion combination. The results showed that except for the DC-tumor hybrids, which play the key role in the antitumor immunity, the non-adherent cell portion, mostly containing unfused DCs, have a large contribution to antitumor immunity. The cytotoxic T lymphocyte (CTL) assays showed that purified cross cells supplemented with the non-adherent cell populace can elicit the most effective lysis. Thus, the unfused DCs should also become taken into account during fusion cross study. We further explored the mechanism of immunogenicity from unfused DC in Lersivirine (UK-453061) non-adherent cell portion. For the first time, we showed that unfused DCs can phagocytose apoptotic/necrotic tumor cells or tumor cell debris and then undergo maturation, which may be the main reason why the non-adherent cell populace consisting of primarily unfused DCs was able to elicit effective antitumor immunity. We further found it.