The results of this translational pre-clinical study demonstrate that a subset of tumor cells in human carcinomas identified as ALDHbright cells are recognized and eliminated in vitro and in vivo by HLA-A2-restricted, ALDH1A188–96 peptide-specific CD8+ T cells. In human tumor xenograft models, we have demonstrated that adoptive transfer of ALDH1A1-specific CD8+ T cells inhibited growth of subcutaneously growing xenografts and experimental-induced lung metastases. In addition, following surgery to remove a primary tumor, this therapy inhibited spontaneous metastases and prolonged survival of mice.
It was recently reported that the ALDH activity in breast cancer stem cells detected by ALDEFLUOR is primarily due to ALDH1A3 expression rather than ALDH1A1 (30
). In our study, however, we have shown that sorted ALDHbright
cells express higher levels of ALDH1A1 mRNA than ALDH1A3 mRNA. Nonetheless, as previously reported (14
), the ALDH1A-specific CD8+
T cells used in this study recognize the ALDH1A188–96
peptide (LLYKLADLI), but not the highly related peptides derived from the ALDH1A2 (LLDKLADLV) and ALDH1A3 (LLHQLADLV) isoforms. Therefore, regardless of which ALDH1 isoform is prevalently expressed, the recognition of ALDHbright
cells by the ALDH1A 88–96
T cells used in this study is independent of ALDH1A3 expression.
Even though ALDH1A1 is expressed by many cell types, it is highly unlikely that ALDH1A1-based immunotherapy would induce toxicity. Normal stem cells, such as hematopoietic stem cells, which express ALDH1A1 but at a lower level than detected in tumors, have been shown not to be recognized by ALDH1A1-specific CD8+
T cells (14
). Furthermore, although ALDH1A1 is expressed by normal hepatocytes, in agreement with the information in the literature, we have shown that these cells express little to no HLA class I antigen (Ag) on their cell surface; as a result, normal hepatocytes are highly unlikely to be recognized by HLA-class I restricted, ALDH1A1-specific CD8+
T cells (31
Presently, there is little information about the recognition of CIC by HLA class I restricted, CD8+
T cell effectors. To the best of our knowledge, only three studies have investigated this subject; two involve gliobastoma multiforme (GBM) stem cells isolated using selective culture conditions and the third involves sorted colon cancer stem cells identified as a side-staining population. Utilizing a non-tumor related, cytomegalovirus (CMV) antigen as a model TA, Brown et al. (34
) demonstrated recognition of CMV-transfected GBM stem cells by CMV pp65 peptide-specific CTL. Recognition of the targets, however, required targets pulsed with exogenous CMV pp65 peptide. This finding suggests that GBM stem cells expressed HLA class I Ag, but required the exogenous peptide to form a sufficient level of HLA class I Ag-peptide complexes for recognition by the cognate CTL. DiTommaso et al (35
) detected defects in HLA class I Ag and APM component expression in the cultured population of GBM stem cells. As a result, recognition of these target cells by autologous anti-tumor CTL required pretreatment with IFNγ to upregulate HLA class I Ag expression and, presumably, HLA class I Ag/TA peptide complexes, a common situation observed in targeting tumor cells with HLA-class I restricted, TA peptide-specific T cell effectors (36
). In the third study, Inoda et al. (38
) showed that colon carcinoma stem cells are sensitive in vitro
and in vivo
to HLA class I-restricted CTL recognizing an epitope derived from the tumor associated centrosomal protein 55kDa protein, CEP55, which is expressed by the tumor initiating cells as well as the bulk population of cells in the colon carcinoma cell lines studied. Since ALDH1A1 is expressed by CIC present in colon carcinomas and gliomas (10
), targeting CIC populations in these tumors with ALDH1A1-specific CD8+
T cells is also possible and should be more selective.
Our results strongly support further development of strategies that would incorporate ALDH1A1-based immunotherapy to target CIC. The constraints of a practical evaluation of a T cell-based immunotherapy using human xenograft mouse models required adoptive transfer of the immune effector cells. Using recombinant DNA or optimized traditional protocols, sufficient numbers of TA-specific T cells can be generated in vitro
for adoptive T-cell based immunotherapy; this strategy has been shown in recent years to yield beneficial clinical responses in subjects with cancer (40
). Nonetheless, the development and implantation of ALDH1A1-based immunotherapy need not preclude a vaccine-based approach.
In accordance with the cancer stem cell theory, the elimination CIC should be the critical criteria used to define the efficacy of a therapy, rather than only reduction in tumor volume. Our research demonstrates for the first time the potential ability of an immunotherapy to achieve the objective of targeting CIC in tumors. However, while we are aware that therapeutic protocols can promote tumor escape, our findings highlight the benefit that T cell-based immunotherapy offers, which combined other independent therapeutic modalities, such as tumor antigen- specific mAb and/or inhibitors of aberrantly regulated stem cell signaling pathways (13
), would minimize the potential of tumor escape.
Tumor cells expressing high levels of aldehyde dehydrogenase (ALDH) have been identified by flow cytometry as ALDHbright cells and shown to have the properties attributed to cancer initiating cells (CIC). CIC are resistant to conventional cancer treatments and considered responsible for recurrence and metastasis. Pertinent to developing immunotherapy for targeting CIC, these cells express ALDH1A1, a tumor associated antigen recognized by HLA class I restricted, CD8+ T cells, which can be induced/generated in vitro and are present in human subjects with cancer.
This study demonstrates that ALDHbright cells are sensitive to cytolysis by ALDH1A1-specific CTL in vitro. In preclinical models of human tumor xenografts growing in immunodeficient mice, adoptive therapy with ALDH1A1-specific CD8+ T cells was shown to target ALDHbright cells and inhibit xenograft growth, metastases or prolong survival. Our results demonstrate the usefulness of ALDH1A1 as a target of T cell-based immunotherapy to eliminate CIC in tumors.