Prostate cancer stem cells are targets of both innate and adaptive immunity and elicit tumor-specific immune responses

Abstract

According to the cancer stem cell (CSC) theory, therapies that do not target the CSC compartment have limited, if any, chances to eradicate established tumors. While cytotoxic T lymphocytes (CTLs) have the potential to recognize and kill single neoplastic cells within a tissue, whether CSCs can be targeted by the immune system during spontaneous or vaccination-elicited responses is poorly defined. Here, we provide experimental evidence showing that CSC lines established from the prostate of transgenic adenocarcinoma of the mouse prostate (TRAMP) mice expressed prostate cancer-associated antigens, MHC Class I and II molecules as well as ligands for natural killer (NK) cell receptors. Indeed, CSC were targets for both NK cell- and CTL-mediated cytotoxicity, both in vitro and in vivo. The administration of dendritic cells pulsed with irradiated CSCs induced a tumor-specific immune response that was more robust than that induced by dendritic cells pulsed with differentiated tumor cells, delayed tumor growth in mice challenged with prostate CSCs and caused tumor regression in TRAMP mice. Thus, CSC are targeted by both innate and adaptive immune responses and might be exploited for the design of novel immunotherapeutic approaches against cancer.

Keywords: cancer stem cells; dendritic cell; prostate cancer; tumor immunity; vaccination.

Figure 1. Phenotypic characterization of cancer stem cells. Prostate adenocarcinoma-derived stem cells (PAC-SCs, left column), prostatic neuroendocrine tumor-derived stem cells (PNE-SCs, middle column) and TRAMP-C1 cells (right column) were harvested, dissociated to single-cell suspension, stained with FITC-, PE- or APC-conjugated antibodies specific for the indicated markers and analyzed by cytofluorimetry. Histograms illustrate the expression of specific markers (black profiles). White profiles represent isotype controls. Each panel is representative of three independent experiments.

Figure 2. Prostate adenocarcinoma-derived and prostatic neuroendocrine tumor-derived stem cell express MHC molecules and other relevant immunologic markers. Prostate adenocarcinoma-derived stem cells (PAC-SCs, left column), prostatic neuroendocrine tumor-derived stem cells (PNE-SCs, middle column) and TRAMP-C1 cells (right column) were cultured in standard medium (black profiles) or in the presence of interferon γ (IFNγ, gray profiles) for 48 h, then harvested and analyzed as described in the legend to Figure 1. Histograms illustrate the expression of specific markers (black or gray profiles). White profiles represent isotype controls. Each panel is representative of three independent experiments.

Figure 3. Prostate adenocarcinoma-derived and prostatic neuroendocrine tumor-derived stem cell express prostate-specific antigens and can be targeted in vitro by antigen-specific cytotoxic T lymphocytes and natural killer cells. (A) Expression of STEAP, PSCA or Tag was measured in prostate adenocarcinoma-derived stem cells (PAC-SCs), prostatic neuroendocrine tumor-derived stem cells (PNE-SCs) or TRAMP-C1 cells by real-time PCR and the ΔΔCT method. Expression data are normalized to TRAMP-C1 (for STEAP and PSCA) or B6/K0 (for Tag) cells. (B) Upper left panel. Tag-specific CD8⁺ T-cell blasts were tested for their cytotoxic activity against PAC-SCs, PNE-SCs, unpulsed (−) or Tag-IV_404–411-pulsed RMA cells (+) in a standard ⁵¹Cr release assay (effector to target ratio of 50:1). Upper right panel, PSCA-specific CD8⁺ T-cell blasts were challenged with PAC-SCs, PNE-SCs, unpulsed (−) or PSCA_83–91-pulsed RMA cells (+) and analyzed for intracellular interferon γ (IFNγ) production by cytofluorimetry. Data are reported as the percentage of CD44⁺IFNγ⁺ cells among CD8⁺ T cells in each experimental condition tested. Values reported in each column are subtracted of background noise (i.e., IFNγ production against the irrelevant target RMA). Lower panels, representative dot plots. Dot plots are gated on CD8⁺ T cells. The percentage of CD44⁺IFNγ⁺ cells is indicated in each plot. (B) Natural killer (NK) (left panel) and lymphokine-activated killer (LAK) (right panel) cells were tested for their cytotoxic activity against PAC-SCs, PNE-SCs, RMA-S (+) or RMA cells (−) in a standard ⁵¹Cr release assay (effector to target ratios of 40:1 or 50:1, in the left and right panel, respectively). (D) 2 × 10⁶ PAC-SCs (left panel) or PNE-SCs (right panel) were injected in 200 μL DMEM into NOD-SCID, nude, wild type (WT) or TRAMP mice (at least 10 mice/group). Mice were monitored for tumor formation and survival, and sacrificed when lesions reached 100 mm² of area or after 6 mo of observation. Data were compared with ANOVA followed by Tukey’s tests: *p < 0.05; **p < 0.01; ***p < 0.001. Each panel is representative of at least two independent experiments.

Figure 4. Vaccination with dendritic cells pulsed with prostate adenocarcinoma-derived or prostatic neuroendocrine tumor-derived stem cells elicit an antigen-specific cytotoxic T-lymphocyte response. (A–C) C57BL/6 mice were injected intradermally with 5 × 10⁵ dendritic cells (DCs) pulsed with prostate adenocarcinoma-derived stem cells (DC+PAC-SC), prostatic neuroendocrine tumor-derived stem cells (DC+PNE-SC), TRAMP-C1 cells (DC+TRAMP-C1), unpulsed DCs (DC w/o) or 200 μL of PBS (naive) and killed one week later. (A) Splenocytes were stimulated in vitro and 5-d-old blasts were assessed for cytotoxic activity against PAC-SCs (black squares), PNE-SCs (black triangles), TRAMP-C1 cells (white circles) or RMA cells (black circles) in standard ⁵¹Cr release assays. (B) Alternatively, blasts were challenged as indicated and analyzed for intracellular interferon γ (IFNγ) production by cytofluorometry. Dot plots are gated on CD8⁺ T cells. The percentage of CD44⁺IFNγ⁺ cells is indicated in each plot. (C) Histograms depict the quantification of IFNγ production by CD8⁺ T cells against PAC-SCs (left panel) PNE-SCs (middle panel) or TRAMP-C1 cells (right panel). Values are subtracted of background noise (i.e., IFNγ production against the irrelevant target RMA). Each panel is representative of at least three independent experiments. Data were compared with Student's t tests: *p < 0.05.

Figure 5. Dendritic cells pulsed with prostate adenocarcinoma-derived stem cells delay the growth of transplantable prostate tumors. (A) C57BL/6 male mice were immunized with 5 × 10⁵ dendritic cells (DCs) pulsed with either prostate adenocarcinoma-derived stem cells (DC+PAC-SC; n = 16, black squares), either prostatic neuroendocrine tumor-derived stem cells (DC+PNE-SC; n = 10, black circles), either TRAMP-C1 cells (DC+TRAMP-C1; n = 10 white triangles) or nothing (DC w/o; n = 14, white squares) and challenged one week later with 2.5 × 10⁶ TRAMP-C1 cells s.c.. Mice were monitored twice a week and sacrificed when the tumor size reached a surface area ≥ 100 mm². Data are reported in a Kaplan–Maier plot. Statistical comparisons were performed by means of the logrank tests: DC+PAC-SC vs DC+TRAMP-C1, p = 0.044 (*); DC+PAC-SC vs DC w/o, p = 0.029 (*); DC+PAC-SC vs DC+PNE-SC, p = 0.024 (*) All the other comparisons were not statistically significant. (B) C57BL/6 mice were immunized either with DC+PAC-SC (n = 5; black squares) or DC w/o (n = 5; white squares), challenged one week later with 7 × 10⁴ RMA cells and monitored as described in (A). (C) C57BL/6 males were challenged with 2 × 10⁶ PAC-SCs admixed with Matrigel^™ s.c. and immunized two weeks later with DC+PAC-SC (n = 10, black squares) or DC w/o (n = 10, white squares). Mice were monitored three times a week for tumor formation and progression, and were killed 78 d after tumor challenge. Data are reported as average ± SD of tumor volume (mm³). Statistical comparisons were performed by means of Student's t-tests: **p < 0.01.

Figure 6. Dendritic cells pulsed with prostate adenocarcinoma-derived stem cells cooperate with hematopoietic stem cell transplantation and donor lymphocyte infusion in inducing tumor regression in TRAMP mice. (A and B) Sixteen week-old TRAMP mice received total body irradiation (TBI), 1 × 10⁷ bone marrow cells from C57BL/6 female donors (hematopoietic stem cell transplantation, HSCT), 6 × 10⁷ splenocytes from non-presensitized C57BL/6 female donors (donor lymphocyte infusion, DLI), two vaccinations with dendritic cells (DCs) pulsed with either prostate adenocarcinoma-derived stem cells (DC+PAC-SC; n = 6), either STEAP (CD+STEAPn = 9) or nothing (DC w/o; n = 6), and were killed one week after the last vaccination. The urogenital apparata (UGA) of euthanized mice were collected and embedded in paraffin. Slides were stained with hematoxylin and eosin (H&E) (B) and scored by a trained pathologist in a blind fashion (A). Statistical comparisons were performed by means of χ² tests: *p < 0.05