A tumor metastasis‐associated molecule TWIST1 is a favorable target for cancer immunotherapy due to its immunogenicity

Abstract Although neoantigens are one of the most favorable targets in cancer immunotherapy, it is less versatile and costly to apply neoantigen‐derived cancer vaccines to patients due to individual variation. It is, therefore, important to find highly immunogenic antigens between tumor‐specific or associated antigens that are shared among patients. Considering the cancer immunoediting theory, immunogenic tumor cells cannot survive in the early phase of tumor progression including two processes: elimination and equilibrium. We hypothesized that highly immunogenic molecules are allowed to be expressed in tumor cells after an immune suppressive tumor microenvironment was established, if these molecules contribute to tumor survival. In the current study, we focused on TWIST1 as a candidate for highly immunogenic antigens because it is upregulated in tumor cells under hypoxia and promotes tumor metastasis, which is observed in the late phase of tumor progression. We demonstrated that TWIST1 had an immunogenic peptide sequence TWIST1140–162, which effectively activated TWIST1‐specific CD4+ T‐cells. In a short‐term culture system, we detected more TWIST1‐specific responses in breast cancer patients compared with in healthy donors. Vaccination with the TWIST1 peptide also showed efficient expansion of TWIST1‐reactive HTLs in humanized mice. These findings indicate that TWIST1 is a highly immunogenic shared antigen and a favorable target for cancer immunotherapy.


| INTRODUC TI ON
As immune checkpoint inhibitors show marked antitumor effects in clinical settings, immunotherapy has been in the spotlight in cancer research. 1  and (4) neoantigens, which are encoded by mutated genes in tumor cells. 7 Because of their high immunogenicity, neoantigens are the most favorable targets in cancer immunotherapy. However, it is less versatile and costly to apply neoantigen-derived cancer vaccines to patients, because epitope sequences vary in individuals. This problem would be resolved if we had highly immunogenic shared or tumor-associated antigens and applied them clinically.
Based on cancer immunoediting theory, 8  Twist is evolutionarily conserved from invertebrates to humans and two Twist genes exist in vertebrates, Twist1 and Twist2. 9 In the late phase of tumor development, tissue oxygen tension is reduced (hypoxia) in the tumor microenvironment, 10 resulting in ectopic TWIST1 expression in a hypoxia-inducible factor (HIF) transcription factor, HIF-2α-dependent manner. 11 Ectopic Twist 1 expression is found in various types of tumors including breast cancer, 12 non-small-cell lung cancer, 13 prostate cancer, 14 gastric cancer, 15 melanoma, 16 osteosarcoma, 17 and hepatocellular carcinoma, 18 and esophageal squamous cell carcinoma. 19 Twist1 promotes transcriptional activity to upregulate N-cadherin and suppress E-cadherin expression and induces epithelial-to-mesenchymal transition (EMT) in cancer cells. 20,21 Therefore, its enhanced expression predicts a poor prognosis in cervical cancer, 22 gastric cancer, 23 ovarian cancer, 24 esophageal squamous cell carcinoma, 25 and chronic kidney disease. 26 Based on those characteristics of TWIST1 showing ectopic expression in late phase of tumor progression, we hypothesized that Twist1 has a high immunogenicity and could be a favorable target for cancer immunotherapy.
In the current study, we found that a TWIST1-derived peptide efficiently expanded TWIST1-specific CD4 + helper T-cells (HTLs), which also responded to TWIST1-expressing tumor cell lines.
TWIST1-specific HTLs were detected more frequently in patients with breast cancer who were positive for TWIST1 compared with healthy donors. Furthermore, vaccination with the TWIST1 peptide also showed efficient expansion of TWIST1-reactive HTLs in humanized mice. These findings suggested that TWIST1 is a highly immunogenic shared antigen and a favorable target for cancer immunotherapy.

| Clinical samples
Tumor tissue samples were obtained from patients with breast cancer by surgical resection at Asahikawa Medical University Hospital.
The characteristics of the nine breast cancer patients tested in the current study are summarized in Table 1. This study was approved by the Research Ethics Committee of Asahikawa Medical University and was performed in accordance with the Declaration of Helsinki.
Written informed consent was obtained from all donors who provided samples.

| In vitro generation of TWIST1-reactive HTL lines
The procedure for the expansion of peptide-specific HTLs has been described in detail previously. 6 Briefly, monocytes and CD4 + T-cells were purified from PBMCs using MACS microbeads for CD14 and CD4, respectively (Miltenyi Biotech). Monocytes were differentiated into DCs using GM-CSF (50 ng/ml) and IL-4 (1000 IU/ml). DCs were pulsed with TWIST1 peptide (3 μg/ml for 3 h at room temperature) and then co-cultured with autologous CD4 + T-cells in 96- We measured the absorption at 450 nm using GloMax Discover Microplate Reader (Promega). AIM-V medium (Invitrogen) supplemented in 3% of human male AB serum (Innovative Research) was used as complete culture medium for all experiments. All blood materials were acquired after informed consent was appropriately obtained.   Bold indicates that its allele-restricted HTLs were generated.

| Quantitative real-time PCR
normalize each mRNA expression level, which was calculated in each experiment using the ΔΔC t method.

| Evaluating frequency of TWIST1-specific T-cells in a short-term culture system
PBMCs (1.5-2 × 10 6 ) of healthy donors and patients with breast cancer were stimulated with the TWIST1 140-162 peptide (10 μg/ml) in the presence of IL-2 (10 IU/ml) in 24-well plates as described previously. 28 Seven days after peptide stimulation, counts of IFNγproducing cells were assessed using ELISPOT assay.

| Immunohistochemistry
Immunohistochemistry (IHC) analysis of breast cancer specimens was performed using the EnVision™ HRP System (K5361, Dako) as described previously. 29 Formalin-fixed sections were obtained from breast cancer patients. Samples were boiled in EDTA buffer (pH 9.0) for antigen retrieval, and endogenous peroxidase activity was inhibited according to the manufacturer's instructions.
Sections were then incubated with mouse anti-human TWIST polyclonal antibody (C-17, Santa Cruz 1:300) overnight at 4°C, followed by incubation with an HRP-conjugated secondary antibody and substrate. Images were acquired using a BZ-X700 microscope (Keyence).

| Statistical analysis
Statistical analysis was performed using GraphPad Prism 9.3.1 (GraphPad Software). Differences between two groups and among multiple groups were analyzed using unpaired t-tests and one-way ANOVA with Tukey's multiple comparison test, respectively. Data are presented as mean ± SD or SE, and a p-value < 0.05 was considered statistically significant.

| High frequency of TWIST1-specific HTLs in the periphery of patients with breast cancer expressing TWIST1
Because we found that TWIST1 140-162 -specific HTL lines responded to endogenous TWIST1-expressing tumor cell lines, we addressed whether TWIST1 was highly immunogenic enough to spontaneously stimulate TWIST1-specific T-cells in the body. Therefore, we stimu-  Table 1). Cesson and colleagues also experienced a similar situation when they analyzed the immunogenicity of MAGE-A3 in cancer patients. 30 In some breast cancer patients BC8 and BC9, the TWIST1 140-162 peptide-specific T-cell response was restricted to HLA-DR, even though there were no specific responses in healthy donors even in the ELISPOT assay ( Figure 3C). These findings suggested that TWIST1 is highly immunogenic and naturally activates TWIST1-specific T-cells in breast cancer patients.
Interestingly, we detected TWIST1 140-162 peptide-specific production of not only IFNγ but also TNFα and granzyme B in the samples from BC5, BC6, and BC7, indicating that they showed a Th1 cell phenotype ( Figure 4). As we had previously demonstrated that granzyme B-producing HTLs directly killed tumor cells, 31,32 these TWIST1-specific HTLs would show antitumor cytotoxicity. These results suggested that the TWIST1 140-162 peptide could effectively activate TWIST1-specific HTLs to enhance cell-mediated immunity against tumors expressing TWIST1/2.

| Highly immunogenic activity of the TWIST1 140-162 peptide in vivo
To address whether the TWIST1 140-162 peptide effectively ex- The function of TWIST1 is still controversial. Recent studies have shown that TWIST1 not only contributes to metastasis of tumor cells through EMT, 34 but also is involved in resistance to chemotherapy. 35 However, in either case, TWIST1 is highly expressed in metastatic tumor cells and therefore is a potential therapeutic target for inhibiting tumor metastasis and treating metastatic tumors. Especially in patients with chemotherapy-resistant tumors, cancer immunotherapy targeting TWIST1 would provide an alternative treatment strategy.

ACK N OWLED G M ENTS
The authors thank Dr. François A Lemonnier the inventor of the HHDII-DR1 mice (Paris University) and Ms. Rie Matsumoto (Asahikawa Medical University) for devotedly maintaining the mice.

D I SCLOS U R E
The authors have no conflict of interest.