ALDH1A1 and ALDH1A3 are expressed by cancer stem cells
High levels of ALDEFLUOR/ALDH activity have been used as a CSC marker in a variety of solid tumors. ALDH1A1 and ALDH1A3 are the ALDH isoforms most commonly expressed in CSCs and are also important functionally for CSCs by enhancing cell growth and reducing apoptosis via degradation of retinoic acid.5 ALDH inhibitor treatments and ALDH1A peptide-specific CD8+ T-cell studies successfully reduced CSCs, tumor growth, and metastasis,5,6 suggesting that ALDHs are promising targets for therapeutic strategies. However, small molecule inhibitors of ALDH1A1 and ALDH1A3 have not shown much success in clinical applications. One of these challenges is the lack of drug solubility. The authors hypothesized that using ALDH1A1 and ALDH1A3 as peptide targets for DC vaccines would be a more effective treatment strategy and that combining this with PD-1/PD-L1 blockade may further enhance the success of this strategy.6
Two peptides were then derived from ALDH1A1 and ALDH1A3. Initial studies characterized in vitro T-cell responses to unloaded DCs (negative control), ALDH1A1 peptide-DCs, ALDH1A3 peptide-DCs, ALDH 1A1+1A3 peptides-DCs, and D5 ALDHhigh CSC lysate-DC (positive control). Enhanced CD3+ T-cell proliferation was observed with all the stimulations compared to the unloaded control. The ALDH 1A1+1A3 peptides-DC stimulated T-cells had the highest of the peptide-DC stimulations and was comparable to the CSC lysate-DC stimulation (31.5% and 35.5% respectively). Similar trends were observed for CD3+ T-cell expansion and cytotoxicity studies.
Since the initial in vitro studies showed promise, the ALDH peptide-DC vaccines were examined in a D5 melanoma tumor model. Two vaccines were injected 14 and 7 days pre tumor cell injection. All the peptide-DC vaccines had decreased tumor volume after 24 days compared to the PBS-treated animals, with the ALDH 1A1+1A3 having the most dramatic impact. T-cells collected from spleens of these immunized mice showed increased cytotoxic activity compared to those isolated from the PBS treated mice. Again, the ALDH 1A1+1A3 had the highest cytotoxicity of the three, likely because these T-cells had the highest level of intracellular IFN-γ and secreted IFN-γ.
After successful induction of T-cell responses by the ALDH peptide-DC vaccines, humoral responses were examined. Mouse IgG ELISA was used to quantify plasma IgG from each of the treated animals. Plasma IgG from the ALDH peptide-DC vaccine-treated mice had increased binding to ALDHhigh D5 CSCs compared to PBS treated mice.
Complement-dependent cytotoxicity assays were performed to examine potential impact of IgG binding. All the ALDH peptide-DC vaccine-primed animals had higher complement-dependent cytotoxicity activity and lysis of ALDHhigh D5 CSCs compared to the PBS control mice. ALDH 1A1+1A3 peptide-DC vaccinated animals had the highest cytotoxicity of the DC-vaccinated mice.
To further characterize the in vivo tumor environment, residual tumors were examined by immunohistochemistry and single cell suspensions were made to quantify CSCs. ALDHhigh CSCs were reduced in all ALDH peptide-DC vaccinated animals, with the dual peptide causing the most significant reduction - from 15% of the tumor composition to less than 2%. IHC assays showed increased CD3+ T-cell infiltration in the ALDH peptide-DC vaccinated mice.