Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death

Abstract

Systemic anticancer chemotherapy is immunosuppressive and mostly induces nonimmunogenic tumor cell death. Here, we show that even in the absence of any adjuvant, tumor cells dying in response to anthracyclins can elicit an effective antitumor immune response that suppresses the growth of inoculated tumors or leads to the regression of established neoplasia. Although both antracyclins and mitomycin C induced apoptosis with caspase activation, only anthracyclin-induced immunogenic cell death was immunogenic. Caspase inhibition by Z-VAD-fmk or transfection with the baculovirus inhibitor p35 did not inhibit doxorubicin (DX)-induced cell death, yet suppressed the immunogenicity of dying tumor cells in several rodent models of neoplasia. Depletion of dendritic cells (DCs) or CD8+T cells abolished the immune response against DX-treated apoptotic tumor cells in vivo. Caspase inhibition suppressed the capacity of DX-killed cells to be phagocytosed by DCs, yet had no effect on their capacity to elicit DC maturation. Freshly excised tumors became immunogenic upon DX treatment in vitro, and intratumoral inoculation of DX could trigger the regression of established tumors in immunocompetent mice. These results delineate a procedure for the generation of cancer vaccines and the stimulation of anti-neoplastic immune responses in vivo.

Figure 1.

In vitro characterization of the different cell death modalities induced in CT26 cells. (A) FACS analysis of cells treated in vitro with the indicated agents as specified in Materials and methods. ZVAD, Z-VAD-fmk. Numbers indicate the percentage of cells (X ± SD, n = 5) in each quadrant. (B) Immunoblot analyses of cells treated as in A. Cellular extracts were subjected to the immunodetection of activated caspase 3 (Casp3a), HMGB-1, and HSP-70. (C) TUNEL staining (green) of cells counterstained with DAPI (blue). The percentage of TUNEL⁺ cells (X ± SEM, n = 3) was determined. (D) Transmission electron microscopy. Representative microphotographs are shown. Apoptotic cells showing chromatin condensation are labeled with an “A.” (E) Cells treated with the indicated agents as in A were washed and plated to determine the frequency of surviving clones, defining the control value of untreated cells as 100%. Results are representative of three independent experiments.

Figure 2.

Immunogenicity of the different cell death types. (A) Tumor evolution of DX- and DXZ-treated cells in nu/nu mice. Cells treated as in Fig. 1 were injected subcutaneously (3 × 10⁶/mouse). (B) Tumor evolution of DX- and DXZ-treated cells in immunocompetent BALB/c. Note that MC- or F/T-treated mice also did not form tumors. (C) Immunogenic effect of DX-treated tumor cells. BALB/c mice were injected into one flank with live tumor cells and into the opposite flank with DX-, DXZ-, MC-, or F/T-treated cells, and the evolution of tumors was monitored. (D) Persistent, specific antitumor immunity elicited by DX-treated cells. Animals immunized with DX-treated CT26 cells that remain tumor-free after 120 d (see lower left panel in C) or age-matched control mice were challenged with CT126 cells or the unrelated TSA tumor cell line. (E) Adoptive transfer of antitumor immunity elicited by DX-treated cells. BALB/c mice were injected intravenously with 10⁷ splenocytes from naive control animals or animals immunized with DX-treated CT26 cells that remain tumor-free (as in B and D) followed by subcutaneous injection of 5 × 10⁵ tumor cells. (F) Effect of the timing of antitumor vaccination. CT26 cells treated as in Fig. 1 were injected at the same time as live tumor cells (simultaneous injection as in C) or were injected once 1 wk before challenge with live tumor cells (prophylactic immunization; n = 15 per group). (G) Replacement of DX by other anthracyclins. Animals were injected with CT26 cells treated with daunorubicin (DA) or idarubicin (ID), instead of DX, or PBS only (CO), and the growth of live CT26 cells simultaneously injected into the opposite flank was monitored as in C. All figures represent three to six independent experiments. *, P < 0.05 versus control (CO). Note that growth curves of tumors are only shown to the level at which ethical guidelines oblige to kill the animals.

Figure 3.

Contribution of DX to the immunogenic nature of apoptotic tumor cells. (A) DX incorporation into CT26 cells. Tumor cells were treated in the absence or presence of DX for 24 h as in Fig. 1, and the incorporation of DX into cells was measured by assessing the DX-specific red fluorescence in the FACS (left). Alternatively, CT26 cells were killed by prolonged culture in MC (as in Fig. 1) followed by the optional addition of DX during the last 15 min of culture, washing, and determination of the DX incorporation (right). (B) Immunogenic effect of DX in vivo. Cells treated as in A (DX, MC, or MC plus DX) were injected subcutaneously in a prophylactic setting (as in Fig. 2 F) 1 wk before challenge with life CT26 cells in the opposite flank followed by monitoring of tumor growth.

Figure 4.

Inhibition of immunogenicity by the caspase inhibitor p35. (A) FACS analysis of CT26 cells transfected with vector only (Neo) or p35 cultured alone or in the presence of DX as in Fig. 1 A. Numbers indicate the percentage of cells (X ± SD, n = 5) in each quadrant. (B) Clonogenic survival of Neo or p35-transfected cells left untreated (100% values) or treated with DX. (C and D) Evolution of CT26 tumors in animals injected simultaneously with Neo-transfected, DX-treated, or p35-transfected DX-treated cells into the opposite flank. Note that only Neo-expressing DX-treated cells confer tumor immunity (P < 0.01).

Figure 5.

Effect of dying tumor cells on DCs. (A and B) In vitro phagocytosis of the DX-, DXZ-, or MC-treated cells (stained with CMTMR) by spleen DCs from Flt3L-injected mice. Representative FACS diagrams are depicted in A, and confocal images of dying tumor cells (red) phagocytosed by purified DC subsets (green) are shown in B. (C) DC maturation of splenic DCs induced by LPS (positive control) and dying or dead CT26 cells. Percentage values in A and C are means of three independent determinations ± SD. (D) Failure of Z-VAD-fmk to inhibit the phagocytosis of DX-treated CT26 cells by DCs. DCs generated as in D were incubated for 90 min with a twofold excess of DX-treated CT26 cells in the presence of the indicated concentrations of Z-VAD-fmk, and the percentage of DCs containing dying or dead CT26 cells was determined as in A. (E) Failure of DX to activate DCs. Bone marrow–derived DCs were activated with LPS as a positive control or with the indicated concentration of DX, and the frequency of apoptotic cells (annexin V⁺) and CD86⁺ cells was determined by immunofluorescence and cytofluorometric analysis. Percentage values in A and C–E are means of three independent determinations ± SD.

Figure 6.

Characterization of the immune effectors induced by dying tumor cells. (A) CTL response of DX- and DXZ-treated cells in immunized mice. Splenic T cells from animals vaccinated with PBS only (CO), DX-treated CT26 cells, or DXZ-treated CT26 cells were tested for their capacity to lyse syngenic cells pulsed with the immunodominant H2L^d- restricted CT26-derived peptide SPSYVYHQF. Representative CTL responses from individual mice are shown on the left, and mean values are shown on the right. R and NR refer to responders and nonresponders, respectively. *, P < 0.01 versus control (CO). (B) Failure of nude mice to mount an immune response against DX-treated CT26 cells. Wild-type or nu/nu BALB/c mice were inoculated with DX-treated CT26 cells 1 wk before the injection of live CT26 cells into the opposite flank, and tumor-free survival was monitored. (C) Requirement of CD8⁺ but not NK cells for the antitumor immune response. Wild-type BALB/c mice received intraperitoneal injections of depleting antibodies specific for CD8 or for the NK marker asialo-GM1 3–4 d before challenge with dying (DX-treated) and live (untreated) CT26 cells into opposite flanks at 1 wk of interval.

Figure 7.

Contribution of specific CTLs and DCs to the immune response against DX-treated tumor cells. (A) Percentages of CD8⁺ lymph node cells expressing TCR that interact with the OVA-derived peptide SIINFEKL presented by H-2K^b 5 d after challenge with either peptide plus adjuvant (P+A) or with DX, DX F/T (cells treated with DX and then F/T), DXZ-, MC-, and F/T-treated B16-OVA cells (three experiments). (B) Absolute number of specific CD8⁺ cells per lymph node determined as in A. Values are X ± SD (n = 3). (C) IFN-γ production as measured after in vitro culture with SIINFEKL in the same experiment. Control values are <50 pg/ml. (D) Effect of the depletion of DCs on the accumulation of specific T cells. Transgenic mice specifically expressing the diphteria toxin (DT) receptor in DCs were pretreated with PBS alone or a dose of diphteria toxin that depletes DCs. The mice were then challenged with DX-treated B16-OVA cells into the food pad. Draining lymph nodes were recovered 48 h later and stained for simultaneous detection of CD8 and OVA peptide–specific T cell receptors. Representative FACS pictograms are shown and values are X ± SD (n = 3).

Figure 8.

Antitumor vaccination with DX-treated melanoma or colon carcinoma cells in distinct rodent models. (A) Percentage of metastasis-free mice observed after intravenous injection of live CT26 cells into animals injected simultaneously with vehicle only (CO) or dying tumor cells generated as in Fig. 1. Untreated controls developed 46 ± 22 metastases (X ± SD, n = 46). (B) Effect of two immunizations with DX-treated CT26 cells 14 and 7 d before intravenous injection of live CT26 cells. Data in A and B have been pooled from three independent experiments. *, P < 0.001 versus control (CO). (C) DX-treated B16A2 cells were injected twice into mice transgenic for HLA.A2 1 and 2 wk before challenge with live B16A2 cells into the opposite flank. *, P < 0.05 versus control (CO). (D) The protocol of simultaneous subcutaneous injection of dying (DX- or DXZ-treated) tumor cells was applied to BDIX rats challenged with PROb cells. *, P < 0.001 versus control (CO). This experiment has been repeated three times, yielding similar results.

Figure 9.

Ex vivo and in vivo induction of immunogenic cell death. (A) Evolution of tumors in mice vaccinated with cells from freshly resected or cryopreserved CT26 tumors, which were left untreated (CO) or treated with DX. (B) Effect of intratumoral chemotherapy on the growth of CT26 colon cancer cells inoculated into immunocompetent BALB/c mice. BALB/c mice previously inoculated with live CT26 cells received intratumoral injection (arrows) of DX, DXZ, or MC as soon as they were palpable ∼14 d after inoculation of tumor cells, and the tumor growth was monitored. Although some DX-treated animals showed a normal tumor growth (n = 12) or a simple delay in tumor growth (n = 12), other animals exhibited stable disease (n = 7) or complete tumor regression (n = 9). (C) Rechallenge of animals cured by intratumoral injection of DX (as in B) or age-matched control (CO) mice with live CT26 cells. Similar results were obtained in three independent experiments. (D) Failure of intratumoral DX injections to confer stable antitumor effects in nu/nu BALB/c mice carrying CT26 tumors. The experiment was performed as in B except that the animals were immunodeficient. (E) Intratumoral injections in the PROb rat colon carcinoma model. Immunocompetent or nu/nu BDIX rats carrying PROb tumors received intratumoral injections of DX, DXZ, or MC, and tumor growth was monitored.