Cancer stem cell vaccination confers significant antitumor immunity

Abstract

Most studies of cancer stem cells (CSC) involve the inoculation of cells from human tumors into immunosuppressed mice, preventing an assessment on the immunologic interactions and effects of CSCs. In this study, we examined the vaccination effects produced by CSC-enriched populations from histologically distinct murine tumors after their inoculation into different syngeneic immunocompetent hosts. Enriched CSCs were immunogenic and more effective as an antigen source than unselected tumor cells in inducing protective antitumor immunity. Immune sera from CSC-vaccinated hosts contained high levels of IgG which bound to CSCs, resulting in CSC lysis in the presence of complement. CTLs generated from peripheral blood mononuclear cells or splenocytes harvested from CSC-vaccinated hosts were capable of killing CSCs in vitro. Mechanistic investigations established that CSC-primed antibodies and T cells were capable of selective targeting CSCs and conferring antitumor immunity. Together, these proof-of-concept results provide a rationale for a new type of cancer immunotherapy based on the development of CSC vaccines that can specifically target CSCs.

©2012 AACR.

Figures

Figure 1

Existence of ALDEFLUOR+ /ALDH+/high populations in murine D5 melanoma and SCC7 squamous cell tumors. The ALDEFLOUR kit labels the ALDEFLUOR positive population including the stem/progenitor cells. The ALDEFLOUR assay isolates the population with a high ALDH enzymatic activity. As negative control, an aliquot of each sample of cells was treated with 50 mmol/l diethylaminobenzaldehyde (DEAB), a specific ALDH inhibitor. To test the purity of the sorted cells, the whole ALDEFLUOR+/ALDH+/high cells (4–6%) and an approximately equal number (5–15%) of the ALDEFLUOR− cells used for the subsequent immunogenicity analyses (ALDH−/low cells) were collected and re-stained with ALDEFLOUR using the same staining protocol. The percentages of ALDH+/high and ALDH−/low cells are listed with the purity of 7 out of the 8 restains being higher than 90%.

Figure 2

Testing of tumorigenicity of ALDEFLUOR+ populations in D5 and SCC7 tumor models. Equal numbers of D5 (A) or SCC7 (B) ALDEFLUOR+ and ALDEFLUOR− cells were injected into the opposite side of the same mouse. Tumor growth was then observed. ALDEFLUOR+ cells can form tumors more efficiently than ALDEFLUOR− cells.

Figure 3

Vaccination of DC pulsed with ALDELFUOR+ D5 cell lysate (CSC-TPDC) induced significantly higher protective immunity against tumor than whole D5 tumor cell lysate pulsed DCs (H-TPDC). (A) CSC-induced protective antitumor immunity in the D5 melanoma model syngenaic to B6 host in a pulmonary metastatic model. The mean numbers of lung metastases (SEM) are depicted in the graph. (B) Pictures of tumors from representative animals in (A). Two of the two independently performed experiments are shown.

Figure 4

CSC-induced protective antitumor immunity was confirmed in the second tumor model (SCC7) syngeneic to a different immunocompetent host (C3H) in a subcutaneous (s.c). tumor setting. The mean tumor sizes (SEM) for the groups are depicted in the graph (A). Data are representative of three experiments performed. (B) Pictures of tumors from representative animals used in (A). (C) Experiments repeating (A) with one additional control group: DCs pulsed with sorted ALDEFLUOR− SCC7 cell lysate (ALDHlow-TPDC). (D) CSC-induced protective antitumor immunity was verified in D5 tumor model with a subcutaneous (s.c). tumor challenge using the ALDEFLUOR+ and ALDEFLUOR− cells isolated from freshly harvested growing D5 tumor.

Figure 5

A. Systemic humoral responses in CSC-TPDC vaccinated immunocompetent host. Splenocytes were harvested from the animals subjected to H-TPDC or CSC-TPDC vaccination, and were activated with anti-CD3/anti-CD28/IL-2 for T cells, or with LPS/anti-CD40 for B cells. The culture supernatants were then collected for IgG detection using ELISA. Data are representative of two experiments performed. B. Plasma harvested from D5 CSC TPDC-treated or SCC7 CSC TPDC-treated animals binds to ALDH+ D5 CSCs and ALDH+ SCC7 CSCs respectively. Data were repeated in a second experiment for the D5 model, and are representative of three experiments performed for the SCC7 model.

Figure 6

Targeting of cancer stem cells by cancer stem cell-primed antibody and complement-dependent cytotoxicity (CDC). Plasma antibody and complement-mediated CSC lysis was assessed by incubating 105 viable ALDH+ CSCs or ALDH− non-CSCs (as control) with plasma harvested from animals subjected to PBS, H-TPDC, or CSC-TPDC treatment in test tubes for 1 h followed by cell culture in the presence of rabbit complement for another hour. Viable cells were then counted under a microscope after trypan blue staining to calculate cell lysis. % of viable cells = viable ALDH+ or ALDH− cells after plasma and complement incubation /105. Lower % of viable cells at the end of incubation indicates more cell lysis. Data of CDC were replicated in a second experiment for both the D5 and SCC7 tumor models.

Figure 7

Targeting of cancer stem cells by cancer stem cell-primed CTLs. Cytotoxicities of CSCs mediated by CTLs generated from the PBMCs (A) harvested from the CSC TPDC-vaccinated animals are shown. The killing of CSCs mediated by the CTLs was measured by an LDH release assay as described in the Materials and Methods. Higher % of cytotoxicity indicates more cell lysis. Data were replicated in a second experiment for both the D5 and SCC7 tumor models. Cytotoxicity of ALDH+ vs. ALDH− D5 cells by effector cells generated from the splenocytes (B) of mice vaccinated with D5 CSC-TPDC was also measured by the LDH release assay as in A. Data are representative of two experiments performed.