Targeting Cancer Stem Cells By Dendritic Cell Vaccine Loaded With Cancer Stem Cell Antigens

Part Ⅰ Identification of cancer stem cells using the stem cell marker ALDH in melanoma and squamous cell carcinomaObjective:Cancer stem cells (CSCs) are defined as a unique subpopulation in tumors that have the ability to initiate tumor growth and sustain tumor self-renewal. Recent evidence suggests that enhanced aldehyde dehydrogenase (ALDH) activity is a hallmark of CSCs measurable by the ALDEFLUOR assay. In this study, we have identified CSCs using stem cell marker ALDH from murine melanoma cell line(D5), murine squamous cell carcinoma cell line(SCC7), murine melanoma and murine squamous cell carcinoma.Method:The ALDEFLUOR kit was used to isolate the population with a high ALDH enzymatic activity. Cells obtained from D5, SCC7, freshly dissociated murine melanoma and murine squamous cell carcinoma. To identify the purity of sorted ALDH+and ALDH-cells, sorted cells were incubated with ALDEFLUOR reagent and analyzed by flow cytometry. In spheroid colony formation assay, sorted cells were inoculated in nonadherent conditions with serum-free media. In vivo, we performed transplantation of sorted cells in mice to test the tumorigenicity. In this assay, mice were monitored for tumor growth. Results:We identified CSC-enriched populations in two different murine tumor models. Two cell lines D5and SCC7had a stable subpopulation of ALDH+cells(approximately4～6%), with the rest (～95%) being ALDH-.The existence of a small percentage of ALDH+cells in established murine tumors was confirmed by analyzing freshly harvested tumor cells. Processed tumor cells from in vivo established D5and SCC7murine tumors also revealed approximately5%of the ALDH+cells. To determine the purity of sorted cells, the whole ALDH+cells and an equal number of the ALDH-cells re-stained with ALDEFLUOR reagent using the same staining protocol. High percentages (>90%) of the ALDH+cells and ALDH-cells after restaining confirmed the purity of originally sorted cells. Sorted D5and SCC7ALDH+cells showed spheroid colony formation in serum-free media in vitro. The tumorigenicity of sorted D5and SCC7ALDH+cells was evaluated in the C57BL/6and C3H hosts, separately. Only ALDH+cells generated tumors while equal numbers or even much greater numbers of ALDH-cells failed to establish tumor.Conclusion:These data indicate that ALDH can serve as a reliable marker for the enrichment of murine D5and SCC7CSCs. It has allowed us to characterize CSC-induced anti-tumor immunity in the immunocompetent murine host in the subsequent experiments. Part Ⅱ Cancer stem cell vaccination confers significant protective anti-tumor immunityObjection:Cancer stem cells (CSCs) are defined as a subpopulation of tumor cells that is resistant to conventional cancer treatments, highly tumorigenic in immunodeficient mice, and responsible for tumor recurrence and metastasis. Thus, the CSC hypothesis promises the development of more effective treatments, aimed not at reducing tumor bulk, but rather at targeting the "beating heart" of the tumor, CSCs. Based on an elevated aldehyde dehydrogenase (ALDH) activity in stem and progenitor cells, ALDH+cells have been identified and isolated from tumors and shown to have characteristics of CSCs. ALDH1A1, belongs to ALDH family, was previously identified as a tumor antigen recognized by CD8+T cells. Based on that information, this study examines the ability of dendritic cell vaccine loaded with ALDH+CSCs antigens to control tumor growth and metastases.Method:To examine the protective antitumor immunity induced by vaccination with DCs pulsed with the lysate of ALDH+cells (ALDH+-TPDC), ALDH+and ALDH" cells were isolated as described above either from cultured D5and SCC7cells or from freshly harvested growing tumors from initial respective D5or SCC7cell injection. ALDH+, ALDH-and unsorted cells were frozen and thaw3times to make cell lysate. Bone-marrow derived DCs were cultured in IL-4and GM-CSF as previously described in our lab, and were pulsed with tumor lysate to generate tumor lysate-pulsed DCs (TPDC). After24hr co-culture, normal animals were vaccinated with ALDH+-TPDC or DCs pulsed with lysate from unsorted heterogeneous tumor cells (H-TPDC), or DCs pulsed with sorted ALDH" cell lysate (ALDH--TPDC) at the same DCs to tumor cell lysate ratio as ALDH+-TPDC. After three times vaccination, the B6mice were challenged with the heterogeneous D5tumor cells i.v and the lungs harvested to enumerate lung metastases. In SCC7model, the C3H mice were challenged with the heterogeneous SCC7tumor cells s.c on the opposite side of the vaccine and the tumor size was monitored.Results:In the D5melanoma i.v tumor model, compared with non-vaccinated, PBS-injected animals (PBS), H-TPDC induced protective immunity against tumor growth. Of note, mice that received ALDH+-TPDC prepared at an identical low lysate to DC ratio resulted in significantly fewer lung metastases than the PBS control group as well as the H-TPDC vaccine group. In the D5melanoma s.c tumor model, the experiments demonstrated that vaccination of DC pulsed with the lysate of ALDH-cells isolated from growing tumor induced significantly higher protective immunity against tumor than H-TPDC as well as DC pulsed with the lysate of ALDH'cells also isolated from growing tumors. However, there was no significant difference between PBS vs. H-TPDC or PBS vs. ALDH--TPDC growth curves. In SCC7s.c models, mice that received ALDH+-TPDC showed significant inhibition of tumor growth compared to the no treatment group (P=0.003), and the tumors were much smaller than those growing in the H-TPDC-treated as well as ALDH"-TPDC hosts. It is worth noting that the p values of the difference between PBS vs. H-TPDC and PBS vs. ALDH--TPDC growth curves using SCC7was<0.05at all time points after tumor inoculation.Conclusion:These data demonstrate ALDH+CSC-plused DC vaccine induced protective immunity against tumor, which control the tumor growth and metastases. It highlights the advantage of using CSCs in vaccination versus the traditional vaccine strategy using bulk unsorted tumor cells or using ALDH-cells. Part III Mechanism of protective anti-tumor immunity induced by cancer stem cell vaccineObjection:Our previous data showed ALDH+CSC-plused DC vaccine induced protective immunity against tumors, which control the tumor growth and metastases. In this study, we explored the possible mechanisms underlying ALDH+CSC-plused DC vaccine induced protective immunity.Method:To test systematic immune responses conferred by ALDH+CSC-based vaccine, spleens were harvested at the end of experiments and spleen B cells were activated with LPS plus anti-CD40. After activation, supernatants were collected and analyzed for IgG production. Plasma was collected from vaccinated hosts at the end of the experiments. IgG level was tested using ELISA. Based on IgG titers, ALDH+cells were incubated with plasma with equal quantity of IgG, and their binding to plasma IgG was detected using flow cytometry. ALDH+CSC lysis mediated by antibodies in plasma was assessed by incubation viable ALDH+CSCs or ALDH-non-CSCs, serving as control, with plasma in the presence of rabbit complement. Viable cells were then counted after trypan blue satining. CTLs were generated from the PBMCs or splenocytes harvested from vaccinated animals by anti-CD3/CD28activation and IL-2expansion. CTL-mediated ALDH+CSC cytotoxicity was tested using the LDH-release assay.Results:It showed that significantly higher IgG production by LPS/anti-CD40activated splenocytes isolated from the animals vaccinated with D5ALDH+-TPDC or SCC7ALDH+-TPDC compared with D5H-TPDC (P=0.004) or SCC7H-TPDC (P=0.031). The immune sera from D5ALDH+-TPDC vaccinated hosts bound to D5ALDH+CSCs (>80%) much more efficiently than the binding of the sera from D5H-TPDC vaccinated hosts or sera from PBS injected hosts to D5ALDH+CSCs (30.6%and30.1%, respectively). Similarly, immune sera from SCC7ALDH+-TPDC vaccinated hosts bound to SCC7ALDH+CSCs (26.4%) significantly more than the binding of the sera from SCC7H-TPDC vaccinated hosts (4.0%) or the background binding by sera from PBS injected hosts (0.9%) to SCC7ALDH+CSCs. To evaluate the immunological significance of the binding of ALDH+CSC-primed antibody to ALDH+CSCs, we examined antibody and complement-dependent cytotoxicity (CDC) of ALDH+CSCs. Immune sera from D5ALDH+-TPDC vaccinated hosts mediated significantly more efficient D5ALDH+CSC lysis than the sera collected from D5H-TPDC vaccinated (P=0.002) or PBS treated (P=0.004) hosts. Similarly, immune sera from SCC7ALDH+-TPDC vaccinated hosts induced significantly SCC7ALDH+CSCs lysis compared with the sera collected from SCC7H-TPDC vaccinated hosts (P=0.001) or from PBS treated hosts (P=0.001), but not the ALDH-SCC7cells. To provide further evidence that the enhanced ALDH+CSC-induced anti-tumor immunity is due to direct targeting of ALDH+CSCs, we harvested PBMCs and splenocytes from D5or SCC7ALDH+-TPDC-vaccinated animals to generate CTLs. The results demonstrated that D5ALDH+-TPDC primed CTLs killed D5ALDH+CSCs efficiently (approximately60%) and significantly more than D5H-TPDC primed CTLs (approximately20%). The killing of ALDH-D5cells by D5ALDH+-TPDC primed CTLs was significantly less effective (approximately20%)(P=0.005). The similar results showed in SCC7model.Conclusion:The results strongly support the potential of ALDH+CSCs-based DC vaccine to specific target ALDH+CSCs in tumors. It provides direct experimental evidence that CSCs can be destroyed by CSC vaccine-primed antibodies and T cells. The immunological targeting of CSCs may provide a novel approach for the development of more effective cancer immunotherapies.