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Despite better control of early-stage disease and improved overall survival of patients with breast cancer, the incidence of life-threatening brain metastases continues to increase in some of these patients. Unfortunately, other than palliative treatments there is no effective therapy for this condition. In this study, we reveal a critical role for Src activation in promoting brainmetastasis in a preclinical model of breast cancer and we show how Src-targeting combinatorial regimens can treat HER2+ brain metastases in this model. We found that Src was hyperactivated in brain-seeking breast cancer cells derived from human cell lines or from patients' brain metastases. Mechanistically, Src activation promoted tumor cell extravasation into the brain parenchyma via permeabilization of the blood--brain barrier. When combined with the EGFR/HER2 dual-targeting drug lapatinib, an Src-targeting combinatorial regimen prevented outgrowth of disseminated breast cancer cells through the induction of cell-cycle arrest. More importantly, this combinatorial regimen inhibited the outgrowth of established experimental brain metastases, prolonging the survival of metastases-bearing mice. Our results provide a rationale for clinical evaluation of Src-targeting regimens to treat patients with breast cancer suffering from brain metastasis. [ABSTRACT FROM AUTHOR]

Background: Tumor-infiltrating lymphocytes (TILs) targeting neoantigens can effectively treat a selected set of metastatic solid cancers. However, harnessing TILs for cancer treatments remains challenging because neoantigen-reactive T cells are often rare and exhausted, and ex vivo expansion can further reduce their frequencies. This complicates the identification of neoantigen-reactive T-cell receptors (TCRs) and the development of TIL products with high reactivity for patient treatment., Methods: We tested whether TILs could be in vitro stimulated against neoantigens to achieve selective expansion of neoantigen-reactive TILs. Given their prevalence, mutant p53 or RAS were studied as models of human neoantigens. An in vitro stimulation method, termed "NeoExpand", was developed to provide neoantigen-specific stimulation to TILs. 25 consecutive patient TILs from tumors harboring p53 or RAS mutations were subjected to NeoExpand., Results: We show that neoantigenic stimulation achieved selective expansion of neoantigen-reactive TILs and broadened the neoantigen-reactive CD4 + and CD8 + TIL clonal repertoire. This allowed the effective isolation of novel neoantigen-reactive TCRs. Out of the 25 consecutive TIL samples, neoantigenic stimulation enabled the identification of 16 unique reactivities and 42 TCRs, while conventional TIL expansion identified 9 reactivities and 14 TCRs. Single-cell transcriptome analysis revealed that neoantigenic stimulation increased neoantigen-reactive TILs with stem-like memory phenotypes expressing IL-7R, CD62L, and KLF2. Furthermore, neoantigenic stimulation improved the in vivo antitumor efficacy of TILs relative to the conventional OKT3-induced rapid TIL expansion in p53-mutated or KRAS-mutated xenograft mouse models., Conclusions: Taken together, neoantigenic stimulation of TILs selectively expands neoantigen-reactive TILs by frequencies and by their clonal repertoire. NeoExpand led to improved phenotypes and functions of neoantigen-reactive TILs. Our data warrant its clinical evaluation., Trial Registration Number: NCT00068003, NCT01174121, and NCT03412877., Competing Interests: Competing interests: NL, SPK and SAR have a pending patent application. The rest of the authors report no competing interest., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Cellular immunotherapies using autologous tumor-infiltrating lymphocytes (TIL) can induce durable regression of epithelial cancers in selected patients with treatment-refractory metastatic disease. As the genetic engineering of T cells with tumor-reactive T-cell receptors (TCRs) comes to the forefront of clinical investigation, the rapid, scalable, and cost-effective detection of patient-specific neoantigen-reactive TIL remains a top priority., Methods: We analyzed the single-cell transcriptomic states of 31 neoantigen-specific T-cell clonotypes to identify cell surface dysfunction markers that best identified the metastatic transcriptional states enriched with antitumor TIL. We developed an efficient method to capture neoantigen-reactive TCRs directly from resected human tumors based on cell surface co-expression of CD39, programmed cell death protein-1, and TIGIT dysfunction markers (CD8 + TIL TP )., Results: TIL TP TCR isolation achieved a high degree of correlation with single-cell transcriptomic signatures that identify neoantigen-reactive TCRs, making it a cost-effective strategy using widely available resources. Reconstruction of additional TIL TP TCRs from tumors identified known and novel antitumor TCRs, showing that at least 39.5% of TIL TP TCRs are neoantigen-reactive or tumor-reactive. Despite their substantial enrichment for neoantigen-reactive TCR clonotypes, clonal dynamics of 24 unique antitumor TIL TP clonotypes from four patients indicated that most in vitro expanded TIL TP populations failed to demonstrate neoantigen reactivity, either by loss of neoantigen-reactive clones during TIL expansion, or through functional impairment during cognate neoantigen recognition., Conclusions: While direct usage of in vitro-expanded CD8 + TIL TP as a source for cellular therapy might be precluded by profound TIL dysfunction, isolating TIL TP represents a streamlined effective approach to rapidly identify neoantigen-reactive TCRs to design engineered cellular immunotherapies against cancer., Competing Interests: Competing interests: FJL, SK, PR, and SAR are listed on an international patent application filed based on the NeoTCR8 signature described in this study. All other authors declare no competing interests., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)