Identification of Common Cancer Antigens Useful for Specific Immunotherapies to Colorectal Cancer and Liver Metastases

Abstract

Stage IV colorectal cancer has a poor prognosis, and liver metastases are prone to recurrence, even after resection. This study aimed to identify common cancer antigens, using immunohistochemical staining, as promising targets for antigen-specific immunotherapies in colorectal cancer. We analyzed expression levels and intracellular localization of seven common cancer antigens, CLDN1, EphB4, LAT1, FOXM1, HSP105α, ROBO1, and SPARC, and human leukocyte antigen (HLA) class I via immunohistochemical staining of 85 surgical specimens from primaries and liver metastases. Staining intensity and positive staining were scored to evaluate antigen expression. In 25 primaries, seven cancer antigens were expressed in 88-96% of cases, while HLA class I was expressed on the cell membrane in 80.0% of cases. In 60 liver metastases, FOXM1 and SPARC expression were approximately half that observed in the primaries. Other antigens and HLA class I were highly expressed in both. Most of the primaries and liver metastases may benefit from chimeric antigen receptor-T cell therapy targeting CLDN1, EphB4, and LAT1. Cases with high HLA class I expression may be suitable for vaccine-based and T cell receptor-T cell therapy targeting CLDN1, EphB4, LAT1, FOXM1, HSP105α, ROBO1, and SPARC for primaries and targeting antigens, excluding FOXM1 and SPARC, for liver metastases.

Keywords: T cell receptor-T (TCR-T) cell therapy; cancer vaccine; chimeric antigen receptor-T (CAR-T) cell therapy; human leukocyte antigen class I; immunohistochemical staining.

Figure 1

The scoring of immunohistochemical staining intensity for common cancer antigens and phenotypes and the scoring of staining intensity for HLA class I. The staining intensity of cancer cells for each of the eight antigens—CLDN1, EphB4, LAT1, FOXM1, HSP105α, ROBO1, SPARC, and HLA class I—was scored as follows: negative: 0, weakly positive: 1, weakly to strongly positive: 2, and strongly positive: 3. Intracellular localization of tumor tissue was evaluated using immunohistochemical analysis. CLDN1, EphB4, and LAT1 were mostly expressed on the cell membrane of cancer cells, FOXM1 was expressed on the cell nuclei, HSP105α and ROBO1 were expressed in the cytoplasm of cancer cells, and SPARC was expressed in the cytoplasm of cancer-associated fibroblasts (CAFs) [31]. For HLA class I expression, the presence and staining of HLA class I were observed in both the cell membrane and cytoplasm, but only staining on the membranes of cancer cells was evaluated.

Figure 2

Heatmap and expression score table of seven common cancer antigens and HLA class I in 14 cases of primary colorectal cancer and liver metastases. The median core values of staining intensity and positive staining for seven common cancer antigens—CLDN1, EphB4, LAT1, FOXM1, HSP105α, ROBO1, and SPARC—and HLA class I on tumor cells from 14 cases each of primary colorectal cancer and liver metastases. The expression levels of each antigen were evaluated, alongside potential T cell therapies (CAR/TCR-T cell therapy) applicable to each case. High expression was defined as a score of >3. Cases with high HLA class I expression were suitable for TCR-T, while those with low HLA class I expression may benefit from CAR-T targeting highly expressed CLDN1, EphB4, and LAT1. The cases were divided into naïve and chemotherapy groups. * The chemotherapy group, including patients who received chemotherapy before or after primary colorectal cancer resection. C: CLDN1, E: EphB4, L: LAT1, F: FOXM1, H: HSP105α, R: ROBO1, S: SPARC, HLA: HLA class I, CAR-T: chimeric antigen receptor-T, TCR-T: T cell receptor-T.

Figure 3

Immunohistochemical staining and analysis of seven common cancer antigens. Two representative cases of 14 patients with primary colorectal cancer and liver metastases are presented. The total expression scores for seven cancer antigens and HLA class I in these two representative cases are displayed in the lower-left corner of each image. Case 6, the patient who received chemotherapy prior to primary and liver metastases resection, exhibited high expression of FOXM1, HSP105α, ROBO1, and SPARC in the primary tumor, whereas HLA class I expression was low. CLDN1 and LAT1 were highly expressed, suggesting potential suitability for CAR-T therapy targeting CLDN1 and LAT1. For the liver metastases of Case 6, most antigens showed high expression, except FOXM1 and SPARC, while HLA class I expression remained low. Case 9 represents the patient who underwent primary tumor resection without prior chemotherapy but received chemotherapy before liver metastasis resection. In this case, the primary tumor showed high expression of HLA class I, CLDN1, LAT1, FOXM1, HSP105α, and SPARC. Hence, this case was deemed suitable for TCR-T therapy targeting CLDN1, LAT1, FOXM1, HSP105α, and SPARC and for CAR-T cell therapy targeting CLDN1 and LAT1. The liver metastases of Case 9 displayed consistent HLA class I expression, but FOXM1 expression was notably decreased compared with the primary tumor, suggesting that TCR-T cell therapy targeting antigens excluding FOXM1, along with CAR-T cell therapy targeting CLDN1, EphB4, and LAT1, may be applicable. * The chemotherapy group, including patients who received chemotherapy before primary CRC or CRCLM resection.

Figure 4

Multiplex fluorescence immunohistochemical (MFIH) staining and analysis of five common cancer antigens and HLA class I. MFIH staining was carried out using specific antibodies: opal 520 (green) for anti-EphB4, opal 540 (blue) for anti-FOXM1, opal 570 (red) for anti-LAT1, opal 620 (orange) for anti-HSP105α, opal 650 (yellow) for anti-HLA class I, and opal 690 (pink) for anti-CLDN1. MFIH results for Case 6 showed low expression of HLA class I in both primary colorectal cancer and liver metastases. CLDN1 and LAT1 were visually confirmed to be highly expressed in primary tumors, with EphB4 also showing high expression in liver metastases along with CLDN1 and LAT1. MFIH analysis of Case 9 visually confirmed uniformly high expression of five cancer antigens and HLA class I in primary tumor. However, FOXM1 expression was reduced in liver metastases. * The chemotherapy group, including patients who received chemotherapy before primary CRC or CRCLM resection.