Background: We have previously demonstrated that hypoxia-activated chimeric antigen receptor (CAR) T-cells could execute robust anti-tumor efficacy with controllable on-target, off-tumor (OTOT) toxicities in the clinical setting. However, some normal tissues or organs, such as bone marrow, rectum, stomach, gallbladder, and epidermis, also exhibit hypoxic microenvironment, which might lead to potential side effects or constraints for the clinical use of hypoxia-responsive CAR T-cells. TGF-β is a significant immunosuppressive factor that impedes immune response in the tumor microenvironment, while its expression in the hypoxic normal tissues is limited. We optimized the construct by introducing an AND-gated circuit complementing the hypoxia-driven conditional activation to reduce OTOT toxicities further. Methods: The AND-gated CAR, BTRPTB001L, consisted of a hypoxia-activated CAR and a constitutively expressing TGF-β chimeric switch receptor (CSR). T-cells isolated from human peripheral blood mononuclear cells were stimulated with anti-CD3/CD28 beads, lentivirally transduced, and expanded ex vivo. CAR/CSR expression was assessed by flow cytometry. IncuCyte was used to analyze the in-vitro cytotoxicity in the presence of TGF-β. The TGF-β sponging capability was assessed by ELISA. Anti-tumor efficacy was evaluated in subcutaneously inoculated xenograft mouse models. Safety evaluation was performed using the AND-gated CAR T-cells targeting the murine orthologous antigen (BTRPTB001L’) in immunocompromised mice. Results: In BTRPTB001L, the fold change of CAR expression level in 1% O2 compared to 21% O2 was 6.33 (P < 0.05), while the CSR expression remained unaffected under either condition. BTRPTB001L showed significantly reduced cytotoxicity under normoxic conditions compared to second-generation (2nd-gen) hypoxia-activated CAR T-cells by co-culturing with target cells. Interestingly, in the presence of TGF-β and under hypoxic conditions, BTRPTB001L showed enhanced cytotoxicity, indicating that the CSR could reverse the inhibitory signal and be specifically activated by the soluble TGF-β in the tumor microenvironment. Notably, the CSR could also sponge TGF-β in the culture supernatant. The improved anti-tumor efficacy of BTRPTB001L was also demonstrated in a xenograft mouse model of pancreatic cancer. Moreover, while the constitutively expressing 2nd-gen CAR recognizing the murine orthologous TAA caused a lethal weight loss phenotype in mice, the BTRPTB001L’-treated group showed no prominent adverse events. Conclusion: BTRPTB001L demonstrated robust anti-tumor potential and improved safety profile compared to hypoxia-responsive 2nd-gen CAR T-cell, supporting its further clinical development. Citation Format: Zhihong Huang, Deping Han, Jean-Paul Thiery, Xi Zhang. Functional enhancement of hypoxia-activated chimeric antigen receptor T-cells by a chimeric switch receptor recognizing TGF-β [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3489.

Tumor-infiltrating lymphocytes (TILs) are heterogeneous populations of lymphocytes residing in tumor tissues, comprising various T-cell clones with specificity towards tumor or viral antigens. TIL therapy has been recently approved for advanced melanoma treatment with high-dose interleukin-2 (IL-2) as a concomitant treatment. However, the accessibility of tumor tissues, the varied abundance and effectiveness of tumor-reactive T-cells, and the toxicity of IL-2 infusion limit the clinical application of TILs toward a broader range of tumor indications. To address the high individual variance, we have engineered TILs expressing chimeric antigen receptor (CAR) constructs targeting glycoproteins on the hepatocellular carcinoma (HCC) cell surface. However, heavy glycosylation of these glycoproteins may mask peptidic epitopes to single-chain antibodies. Previous studies have revealed that the glucose/mannose analog, 2-deoxy-D-glucose (2-DG) treatment of pancreatic cancer cells can disrupt the synthesis of N-glycan moieties on the cell surface and enhance CAR T-cell activity. 2-DG treatment of T cells augmented antitumor activity and cell surface retention of IL-2R of T cells. In this study, we investigated whether the anti-tumor function of CAR-TILs against HCC lines would also benefit from the 2-DG treatment. TILs were isolated from fresh or cryopreserved tumor biopsy samples and stimulated with anti-CD3/CD28 antibodies. Thereafter, TILs were lentiviral transduced with Glypican 3 (GPC3) CAR and membrane bound-interleukins, expanded with γ-irradiated feeder cells in an optimized rapid expansion process (REP), and cryopreserved until use. Phenotypic analyses were performed by flow cytometry. HCC lines were pre-treated with or without 2-DG, and the antigen recognition by antibodies targeting GPC3, CD133, and CD44v6 was evaluated by flow cytometry. In vitro effector function was assessed by IFN-γ release (ELISA) and killing assay (Incucyte). The anti-tumor efficacy of CAR-TILs with or without 2-DG treatment was further assessed in the immunocompromised xenograft model. Pre-treatment of HCC cell lines with 2-DG (1∼10 mM) enhanced the antibody recognition of GPC3 in a dose-dependent fashion while showing no improvement in mAb binding towards CD133/CD44v6. CAR-TILs showed enhanced killing and more IFNγ release towards 2-DG pre-treated GPC3+ HCC lines. The synergistic effect of 2-DG with CAR-TILs was further validated in the immunocompromised murine model, and no apparent toxicity was observed. The proliferation and the CD4/CD8 ratio of CAR-TILs were not affected by 2-DG treatment. 2-DG sensitized GPC3 antigen recognition, thus boosting CAR-TIL cytotoxic activity against GPC3+ HCC cells. These data prompt further investigation before pursuing a combination therapy. Mingyu Liu, Deping Han, Jean-Paul Thiery, Xi Zhang. 2-deoxy-D-glucose sensitizes the antigen recognition of chimeric antigen receptor-expressing tumor-infiltrating lymphocytes toward hepatocellular carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 873.

Transforming growth factor (TGF)-β1 restricts the expansion, survival, and function of CD4+ T cells. Here, we demonstrate that CD4+ but not CD8+ anti-TGF-β CAR T cells (T28zT2 T cells) can suppress tumor growth partly through secreting Granzyme B and interferon (IFN)-γ. TGF-β1-treated CD4+ T28zT2 T cells persist well in peripheral blood and tumors, maintain their mitochondrial form and function, and do not cause in vivo toxicity. They also improve the expansion and persistence of untransduced CD8+ T cells in vivo. Tumor-infiltrating CD4+ T28zT2 T cells are enriched with TCF-1+IL7R+ memory-like T cells, express NKG2D, and downregulate T cell exhaustion markers, including PD-1 and LAG3. Importantly, a combination of CD4+ T28zT2 T cells and CD8+ anti-glypican-3 (GPC3) or anti-mesothelin (MSLN) CAR T cells exhibits augmented antitumor effects in xenografts. These findings suggest that rewiring TGF-β signaling with T28zT2 in CD4+ T cells is a promising strategy for eradicating solid tumors.

Illustrating the hypothesis on the impact of brain sEVs on education and adaptation of breast carcinoma cells outside and then inside the brain microenvironment. Figure created with https://Biorender.com.

Patients with head and neck squamous cell carcinoma (HNSCC) are at a high risk of developing recurrence and secondary cancers. This study evaluates the prognostic and surveillance utilities of circulating tumour cells (CTCs) in HNSCC. A total of 154 HNSCC patients were recruited and followed up for 4.5 years. Blood samples were collected at baseline and follow-up. CTCs were isolated using a spiral microfluid device. Recurrence and death due to cancer were assessed during the follow-up period. In patients with HNSCC, the presence of CTCs at baseline was a predictor of recurrence (OR = 8.40, p < 0.0001) and death (OR= ∞, p < 0.0001). Patients with CTCs at baseline had poor survival outcomes (p < 0.0001). Additionally, our study found that patients with CTCs in a follow-up appointment were 2.5 times more likely to experience recurrence or death from HNSCC (p < 0.05) prior to their next clinical visit. Our study highlights the prognostic and monitoring utilities of CTCs’ in HNSCC patients. Early identification of CTCs facilitates precise risk assessment, guiding treatment choices and ultimately enhancing patient outcomes.

Resistance to DNA-damaging agents is a major unsolved challenge for breast cancer patients undergoing chemotherapy. Here, we show that elevated expression of transcriptional repressor GATA binding 1 (TRPS1) is associated with lower drug sensitivity, reduced response rate, and poor prognosis in chemotherapy-treated breast cancer patients. Mechanistically, elevated TRPS1 expression promotes hyperactivity of DNA damage repair (DDR) in breast cancer cells. We provide evidence that TRPS1 dynamically localizes to DNA breaks in a Ku70- and Ku80-dependent manner, and that TRPS1 is a new member of the DDR protein family. We also discover that the dynamics of TRPS1 assembly at DNA breaks is regulated by its reversible PARylation in the DDR, and that mutations of the PARylation sites on TRPS1 lead to increased sensitivity to chemotherapeutic drugs. Taken together, our findings provide new mechanistic insights into the DDR and chemoresistance in breast cancer patients and identify TRPS1 as a critical DDR protein. TRPS1 may also be considered as a target to improve chemo-sensitization strategies and, consequently, clinical outcomes for breast cancer patients.

Epithelial-mesenchymal transition (EMT) and its reverse mechanism, mesenchymal-epithelial transition (MET), are evolutionarily conserved mechanisms initially identified in studies of early metazoan development. EMT may even have been established in choanoflagellates, the closest unicellular relative of Metazoa. These crucial morphological transitions operate during body plan formation and subsequently in organogenesis. These findings have prompted an increasing number of investigators in biomedicine to assess the importance of such mechanisms that drive epithelial cell plasticity in multiple diseases associated with congenital disabilities and fibrosis, and, most importantly, in the progression of carcinoma. EMT and MET also play crucial roles in regenerative medicine, notably by contributing epigenetic changes in somatic cells to initiate reprogramming into stem cells and their subsequent differentiation into distinct lineages.

RNA interactomes and their diversified functionalities have recently benefited from critical methodological advances leading to a paradigm shift from a conventional conception on the regulatory roles of RNA in pathogenesis. However, the dynamic RNA interactomes in adenoma-carcinoma sequence of human CRC remain unexplored. The coexistence of adenoma, cancer, and normal tissues in colorectal cancer (CRC) patients provides an appropriate model to address this issue. Here, we adopted an RNA in situ conformation sequencing technology for mapping RNA-RNA interactions in CRC patients. We observed large-scale paired RNA counts and identified some unique RNA complexes including multiple partners RNAs, single partner RNAs, non-overlapping single partner RNAs. We focused on the antisense RNA OIP5-AS1 and found that OIP5-AS1 could sponge different miRNA to regulate the production of metabolites including pyruvate, alanine and lactic acid. Our findings provide novel perspectives in CRC pathogenesis and suggest metabolic reprogramming of pyruvate for the early diagnosis and treatment of CRC.

Typical actin cytoskeleton organization observed in most NK cell-conjugated MCF-7 cells. The cell conjugate was acquired on a LSM880 laser scanning confocal microscope (Carl Zeiss) with Airyscan in the high-resolution mode, and the 3D projection was assembled and animated using Zeiss Zen Software. Then NK cell was stained in red (PKH26) whereas the LifeAct-mEGFP-expressing tumour cell appears in green.

MEK inhibition improves in vivo efficacy of cisplatin in SCC xenografts.

Cisplatin resistance in head and neck squamous cell carcinoma (HNSCC) cells is associated with activation of MAPK/ERK pathway.

Src phosphorylation does not mediate MAPK/ERK signaling in lung SCC cells.

Protein-based cancer therapies are considered an alternative to conventional anticancer regimens, providing multifunctional properties while showing low toxicity. However, its widespread use is limited by absorption and instability issues, resulting in higher dosage requirements and a prolonged onset of bioactivity to elicit the desired response. Here, we developed a non-invasive antitumor treatment using designed ankyrin repeat protein (DARPin)-anticancer protein-conjugate that specifically targets the cancer biomarker, epithelial cell adhesion molecule (EpCAM). The DARPin-anticancer proteins bind to EpCAM-positive cancer cells and improve the in vitro anticancer efficacy by over 100-folds within 24 h, where the DARPin-tagged human lactoferrin fragment (drtHLF4) IC50 value is within the nanomolar range. Orally administered drtHLF4 was readily absorbed into the systemic flow of the HT-29 cancer murine model, exerting its anticancer effect on other tumors in the host body. Orally administered drtHFL4 cleared HT29-colorectal tumors using a single dose, whereas intratumoral injection cleared HT29-subcutaneous tumors within three doses. This approach addresses the limitations of other protein-based anticancer treatments by providing a non-invasive anticancer therapy with improved potency and tumor-specificity.

Abstract N6-Methyladenosine (m6A) is the most prevalent internal modification of mammalian mRNAs. Recent studies have shown that m6A methyltransferases METTL3 and METTL14 play important roles in urothelial bladder carcinoma (BLCA). To provide a more comprehensive understanding of the m6A regulatory landscape in bladder cancer, we investigated the role of YTHDF2, a crucial m6A reader, in BLCA. YTHDF2 was frequently upregulated at both the RNA and protein level in BLCA. Functionally, YTHDF2 promoted the proliferation and tumor growth of BLCA cells in vitro and in vivo, respectively. Integrative RNA sequencing and m6A sequencing analyses identified RIG-I as a downstream target of YTHDF2. Mechanistically, YTHDF2 bound to the coding sequence of DDX58 mRNA, which encodes RIG-I, and mediated its degradation in an m6A-dependent manner. Knockdown of RIG-I inhibited apoptosis and promoted the proliferation of BLCA cells. Depleting RIG-I was also able to reverse the effects of YTHDF2 deficiency. YTHDF2-deficient BLCA cells implanted orthotopically in recipient mice activated an innate immune response and promoted recruitment of CD8+ T lymphocytes into the tumor bed and the urothelium. Moreover, YTHDF2 deficiency enhanced the efficacy of Bacillus Calmette-Guérin immunotherapy treatment. This study reveals that YTHDF2 acts as an oncogene in BLCA. YTHDF2 inhibits RIG-I to facilitate immune evasion, supporting testing YTHDF2 inhibition in combination with immunotherapy. Significance: YTHDF2 regulates RIG-I–mediated innate immune signaling to support bladder cancer progression, highlighting the functional importance of m6A modifications in bladder cancer and uncovering therapeutic opportunities to improve patient outcomes.

Supplementary Figure S1. Background information for lung adenocarcinoma cell lines.

Supplementary Figure 1: EMT mediated lorlatinib resistance (corresponding to main Figure 2).