Fig 2: Anti-tumor activity of mCART against LUAD was explored by LDH release assay in vitro. a The tumor-inhibitory rate of mCART in the H1299 and PC9 (both MAGE-A1 positive) cell lines was progressively upregulated along with the increase in the E:T ratio of mCART. The 20:1 ratio of mCART showed the most effective cell killing activity. In comparison, mCART was barely able to kill MAGE-A1-negative cell lines (HBE and PC (shMAGE)). *Significant difference in tumor-inhibitory rate in the mCART group compared with the T group. b A fixed 10:1 E:T ratio of mCART also demonstrated significant tumor-inhibitory efficacy in all MAGE-A1-positive LUAD cell lines. For all the cell viability assays, unrelated-CART and T showed no cell-killing activities, regardless of the E:T ratio selected or the cell type used.*Significant difference in tumor-inhibitory rate in the mCART group compared with the T group. c and d IFN-γ and IL-2 expression were detected when mCART was co-incubated with LUAD cells. The 10:1 E:T ratio of mCART was co-cultured with four different cell lines. After culturing, a larger amount of IFN-γ and IL-2 was released by mCART, and their release was highly associated with the level of MAGE-A1 expression in the LUAD cells. In contrast, the release of IFN-γ and IL-2 remained unchanged in unrelated-CART and T cells. *Significant difference in IFN-γ and IL-2 expression in the mCART group compared with the T group

Fig 3: The detection of MAGE-A1 expression in normal human tissues. a GTEx Portal database illustrated the expression mode of MAGE-A1 (red box) in normal human tissue, and the data showed that MAGE-A1 was mostly expressed in human testis. b IHC analysis in normal human TMA demonstrated that the MAGE-A1 expression was largely witnessed in human testicle samples (red box) while rarely observed in other human tissue samples, including the artery, bladder, brainstem, cerebellum, colon, duodenum, epityphlon, esophagus, ileum, jejunum, liver, lung, medulla, myocardium, pancreas, prostate, skin, spleen, stomach, telencephalon, thyroid, tongue, and trachea

Fig 4: Bioinformatics analyses for the CTA screening. a Raw data were retrieved, and a heat map of the expression of 1019 CTAs in LUAD was created. b A total of 77 candidate CTAs were screened based on score ranking (normalized expression fold > 3%). c In total, 49 candidate CTAs that were exclusively expressed in the testis were screened (GTEx Portal database). d Four CTAs (MAGE-A1, ADAM2, TEX101, and Clorf49) that were expressed in the cytomembranes of cancer cells (expression confidence > 3) were screened (GeneCard database). e Two CTAs (MAGE-A1 and TEX101) had elevated RNA expression in LUAD tissues compared with the corresponding noncancerous tissues (expression fold change > 10, marked by a red box) and were selected (TCGA database). f One CTA (MAGE-A1) that was positively expressed in LC was screened and is marked by a red box (Human Protein Atlas database). g The screening diagram summarizes the entire process by which MAGE-A1 was finally identified as an appropriate CTA from among the original 1019 CTAs

Fig 5: The detection of MAGE-A1 expression in LUAD tissues. a, b qPCR and WB tests in five LUAD and non-cancerous tissue samples showed that the expression of MAGE-A1 in LUAD was elevated compared with that in non-cancerous tissues. *Significant difference of MAGE-A1 expression in LUAD tissue samples compared with non-cancerous tissue samples. p < 0.05. c Detection of MAGE-A1 expression in a tissue microarray (TMA) containing 92 LUAD samples by immunohistochemistry (IHC) analysis. Positive staining of MAGE-A1 was mainly located in the cytoplasm of LUAD cells. d The survival analysis and Kaplan-Meier curve illustrated that positive MAGE-A1 expression (p = 0.022), positive lymph node metastasis (p = 0.001), positive N status (p = 0.002), and advanced TNM stage (p = 0.001) were significantly correlated with a poor prognosis of patients with LUAD