Targeting cancer stem cells with dendritic cell vaccines in melanoma

Efficacy of an ALDH peptide-based dendritic cell vaccine targeting cancer stem cells

Cancer stem cells and aldehyde dehydrogenases

Cancer stem cells (CSCs) make up a distinct tumor cell population with high self-renewal, multiple differentiation capacity, tumorigenesis, and drug resistance.¹ These cells are often the culprit for metastasis and tumor recurrence. Aldehyde dehydrogenases (ALDHs) are important metabolic enzymes in CSCs. These enzymes regulate retinoic acid, reactive oxygen species, and reactive aldehyde levels, all of which can impact cellular processes in CSCs. ALDHs are becoming an accepted marker of CSCs. Additionally, high expression of ALDHs has been correlated with poor prognosis for cancer patients.^2,3

Targeting cancer stem cells with dendritic cell vaccines

Previous work from the authors isolated CSCs from bulk tumors for the generation of lysates that were used to prime DCs and develop DC-based vaccines. These vaccines conferred anti-CSC immunity which prevented metastasis of melanoma to the lungs and subcutaneous tumor growth of head and neck squamous cell cancer in murine models.⁴ While successful in the murine model, the methodology from these studies limits translational feasibility. Instead, the researchers decided to see if similar results could be obtained using shared CSC antigens rather than CSC lysate.

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. ALDH^high 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.

Combinatorial effects of ALDH peptide-DC vaccines and PD-1/PD-L1 blockade

The researchers were then curious as to whether PD-1/PD-L1 blockade would enhance the efficacy of the ALDH peptide-DV vaccines. For this study, mice were inoculated with tumor cells and after 24 hours the ALDH 1A1+1A3 peptides-DC vaccine was administered once a week for two weeks and anti-PD-L1 monoclonal antibody (mAb) or control was administered three times following each vaccination for a total of six treatments in two weeks. As observed previously, the ALDH 1A1+1A3 peptide-DC vaccine reduced tumor volume and the addition of the PD-L1 mAb enhanced the inhibition of tumor growth. The dual peptide-DC vaccine combined with the PD-L1 blockade had increased cytotoxicity against the ALDHhigh CSCs and increased IFN-γ secretion providing a potential mechanism for the reduction in tumor growth. This double treatment group also had increased CD3+ tumor-infiltrating lymphocytes and decreased numbers of ALDH^high CSCs.⁶

The development of cancer vaccines targeting CSCs provides an opportunity to target the cancer cell population most responsible for tumor growth and progression. ALDHs are highly expressed on CSCs across tumor types and therefore provide an opportunity to specifically target these cells with DC-based vaccination.