The progression of cell death affects the rejection of allogeneic tumors in immune-competent mice - implications for cancer therapy

Abstract

Large amounts of dead and dying cells are produced during cancer therapy and allograft rejection. Depending on the death pathway and stimuli involved, dying cells exhibit diverse features, resulting in defined physiological consequences for the host. It is not fully understood how dying and dead cells modulate the immune response of the host. To address this problem, different death stimuli were studied in B16F10 melanoma cells by regulated inducible transgene expression of the pro-apoptotic active forms of caspase-3 (revCasp-3), Bid (tBid), and the Mycobacterium tuberculosis-necrosis inducing toxin (CpnTCTD). The immune outcome elicited for each death stimulus was assessed by evaluating the allograft rejection of melanoma tumors implanted subcutaneously in BALB/c mice immunized with dying cells. Expression of all proteins efficiently killed cells in vitro (>90%) and displayed distinctive morphological and physiological features as assessed by multiparametric flow cytometry analysis. BALB/c mice immunized with allogeneic dying melanoma cells expressing revCasp-3 or CpnTCTD showed strong rejection of the allogeneic challenge. In contrast, mice immunized with cells dying either after expression of tBid or irradiation with UVB did not, suggesting an immunologically silent cell death. Surprisingly, immunogenic cell death induced by expression of revCasp-3 or CpnTCTD correlated with elevated intracellular reactive oxygen species (ROS) levels at the time point of immunization. Conversely, early mitochondrial dysfunction induced by tBid expression or UVB irradiation accounted for the absence of intracellular ROS accumulation at the time point of immunization. Although ROS inhibition in vitro was not sufficient to abrogate the immunogenicity in our allo-immunization model, we suggest that the point of ROS generation and its intracellular accumulation may be an important factor for its role as damage associated molecular pattern in the development of allogeneic responses.

Keywords: DAMPs; ROS; apoptosis; cancer; caspase-3; immunogenicity; necrosis; tBid.

Figure 1

Conditional expression of death inducing proteins. (A) Schematic overview of the constructs used to establish the regulatory system. The vector pWHE644 represents the regulator construct. A human EF1α promoter constitutively transcribes a tricistronic mRNA. This mRNA contains the reverse transactivator rtTA2^S-M2 (blue arrow), the transsilencer tTS^D-PP (yellow arrow), and a selection marker (puromycin resistance; gray arrow). Translation of the latter two genes is mediated by internal ribosome entry sites (IRES; open boxes) from polio-virus (PV) and encephalomyocarditis- virus (EMCV). The vector pWHE655 contains the response unit used for stable transfections. It features the target gene (red arrow) driven by the Tet-responsive promoter TRE_tight (open box, broken arrow) and flanked by two repeats each of a 250 bp sequence from the chicken HS4 insulator (blue triangles). A murine phosphoglycerate kinase 1 promoter (PGK; broken arrow) drives expression of a gene mediating G418-resistance. PolyA sites in all vectors are marked by a “ ⊥.” (B) Schematic representation of the cytotoxic test proteins. The residues that border the active domains expressed in the experiment are indicated above their respective closed box. A methionine added to allow translation is represented by a star. (C) Schematic overview of the regulatory system. In the OFF-State, a transsilencer (white) binds to the minimal promoter (open boxes, broken arrow) and actively suppresses transcription (cross). In the ON-State, doxycycline (blue circles) binds to both transsilencer and reverse transactivator (black). The former dissociates from, the latter binds to the minimal promoter and activates transcription (gray arrow). (D) Response of the regulatory system to different doxycycline concentrations. The B16F10-tBid transfected cell line was incubated for 24 h with various concentrations of doxycycline and mortality was measured, shown for one representative experiment out of three performed. Concentrations between 5 and 10 μg/ml showed the highest extent of cell death. An additional control at 10 μg/ml Doxy with the parental stably transfected cell line B16F10-644 was included to discard doxycycline toxicity at higher concentrations as cause of cell death (dark green diamond). Cell viability at time point “0” is shown as light green diamond.

Figure 2

Six parameter classification by flow cytometry of the cell death phenotype of dying and dead B16F10 cells. Cell death analysis is based on morphological features (FSc and SSc), on the exposure of PS (annexin A5-FITC) and plasma membrane ion selectivity (PI), on the mitochondrial membrane potential [DiIC1(5)] and on nuclear DNA content (Hoechst 33342) detected by flow cytometry. Note: after proper gating, up to eight physiologically different subpopulations can be recorded. Dot plots exemplarily show B16F10-revCasp-3 cells after 18 h of doxycycline (5 μg/ml) treatment (A). Rapid cell death occurred after 6 h in tBid-expressing cells and more than 95% cell death was observed after 24 h. In the presence of various caspase inhibitors [z-VAD-fmk, z-DEVD-fmk (caspase-3 inhibitor) and Ac-LEHD-cmk (caspase-9 inhibitor); all 50 μM], a significant increase in the stressed cell fraction displaying low-mitochondrial potential was observed (B). Expression of revCasp-3 in B16F10 cells induced cell death after 24 h in more than 80% of the cells. z-VAD-fmk (50 μM) completely inhibited doxycycline-driven apoptosis. Note: stressed cells do not arise in this type of cell death induction (C). Expression of CpnT_CTD induced cell death in more than 90% of the cells after 18 h. Note: primary necrosis was the most common type of cell death observed and death occurred independently of caspase activity (50 μM z-VAD-fmk) (D). Lethal UVB irradiation (240 mJ/cm²) of parental B16F10 cells causes a rather slow progressing kind of cell death. Note: in the presence of z-VAD-fmk (50 μM), a significant increase of the stressed cell fraction displaying low-mitochondrial potential was observed (E). Heat shock (56°C, 30 min) caused immediate necrosis in 100% of cells independent of caspase activity (F). Displayed are the mean values from three independent experiments of relative percentages of each cell phenotype during 48 h of culture (B–F).

Figure 3

Reactive oxygen species (ROS) production by dying B16F10 melanoma cells. Cells were induced to die by conditional expression of the death proteins tBid, revCasp-3, and CpnT_CTD or by UVB irradiation, stained with the ROS sensor DCFH and with PI and analyzed by flow cytometry (A). Inhibition of ROS production was performed by treatment with N-acetyl-cysteine (NAC) or mitoTEMPO, 100 μM, respectively, and recorded at 9 h after death induction (B). Mean and SEM values of the mean fluorescence intensities of FL1 in viable cells (PI-negative) are displayed for different time points. At least three independent experiments were performed (Two and one stars indicate statistical significance at the p < 0.001 and p < 0.05 levels, respectively).

Figure 4

Growth of B16F10 melanoma cells in the allogeneic host and concomitant immunity. Four million viable B16F10 cells (VC) were implanted s.c. in the right flank of BALB/c mice. Mice developed tumors reaching their maximum size after 2–3 weeks, followed by rejection [(A), black line]. Mice implanted with 4 million UVB-irradiated cells did not develop primary tumors [(A), purple line]. After challenge with 2 million viable cells s.c. on the left flank, those mice bearing primary tumors did not develop secondary tumors [(B), black line], while mice primarily inoculated with irradiated cells developed tumors similar to those of the naïve group [(B), purple and green lines, respectively]. Mean values (n = 8) and the SEM are displayed. Time points showing statistical significance when compared to the group of mice implanted with VC are highlighted. Two stars and one star indicate statistical significance at the p < 0.01 and p < 0.05 levels, respectively. The two way ANOVA test corrected by Bonferroni was applied in this experiment.

Figure 5

Immune response against dead or dying allogeneic tumor cells. BALB/c mice (n = 5) were immunized in the right flank s.c. (single dose) with B16F10 dying/dead cells. After 10 days, mice were challenged s.c. in the left flank with 2 million viable cells of the parental cell line B16F10-644. Tumor growth was monitored for 30 further days (A). Cell death was induced by UVB irradiation; heat shock; doxycycline-controlled expression of death proteins tBid, revCasp-3, and CpnT_CTD. Displayed are the mean values (n = 5) of relative tumor volumes and SEM [(B), *p < 0.05 after Mann–Whitney U test] and the integral of tumor size [(C), total tumor mass]. Inverse association between ROS production and total tumor mass developed in the allogeneic host (D).