Distant metastasis remains the major cause of morbidity for breast cancer. Individuals with liver or brain metastasis have an extremely poor prognosis and low response rates to anti-PD-1/L1 immune checkpoint therapy compared to those with metastasis at other sites. Therefore, it is urgent to investigate the underlying mechanism of anti-PD-1/L1 resistance and develop more effective immunotherapy strategies for these patients. Using single-cell RNA sequencing, a high-resolution map of the entire tumor ecosystem based on 44 473 cells from breast cancer liver and brain metastases is depicted. Identified by canonical markers and confirmed by multiplex immunofluorescent staining, the metastatic ecosystem features remarkable reprogramming of immunosuppressive cells such as FOXP3+ regulatory T cells, LAMP3+ tolerogenic dendritic cells, CCL18+ M2-like macrophages, RGS5+ cancer-associated fibroblasts, and LGALS1+ microglial cells. In addition, PD-1 and PD-L1/2 are barely expressed in CD8+ T cells and cancer/immune/stromal cells, respectively. Interactions of the immune checkpoint molecules LAG3-LGALS3 and TIGIT-NECTIN2 between CD8+ T cells and cancer/immune/stromal cells are found to play dominant roles in the immune escape. In summary, this study dissects the intratumoral heterogeneity and immunosuppressive microenvironment in liver and brain metastases of breast cancer for the first time, providing insights into the most appropriate immunotherapy strategies for these patients.

The reliability of a handheld myotonometer when used in a clinical setting to assess paraspinal muscle mechanical properties is unclear. This study aimed to investigate the between-session intra-rater reliability of a handheld myotonometer in young adults with low back pain (LBP) in a clinical environment. One assessor recorded lumbar paraspinal muscle tone and stiffness in an outpatient department on two occasions. The intraclass correlation coefficient (ICC), standard error of measurement (SEM), smallest real difference (SRD) and Bland-Altman analysis were conducted to assess reliability. The results indicated acceptable between-days intra-rater reliability (ICC > 0.75) for all measurements. The SEM of the muscle tone and stiffness measurements ranged between 0.20–0.66 Hz and 7.91–16.51 N/m, respectively. The SRD was 0.44–1.83 Hz for muscle tone and 21.93–52.87 N/m for muscle stiffness. SEM and SRD at L1-L2 were higher than those at other levels. The magnitude of agreement appeared to decrease as muscle tone and stiffness increased. The myotonometer demonstrated acceptable reliability when used in a clinical setting in young adults with chronic LBP. Measurements of the upper lumbar levels were not as reliable as those of the lower lumbar levels. The crural attachment of the diaphragm at L1 and L2 may affect paraspinal muscle tone and stiffness during respiratory cycles.

Intrinsic and acquired anti-HER2 resistance remains a major hurdle for treating HER2-positive breast cancer. Using genome-wide CRISPR/Cas9 screening in vitro and in vivo, we identify FGFR4 as an essential gene following anti-HER2 treatment. FGFR4 inhibition enhances susceptibility to anti-HER2 therapy in resistant breast cancer. Mechanistically, m6A-hypomethylation regulated FGFR4 phosphorylates GSK-3β and activates β-catenin/TCF4 signaling to drive anti-HER2 resistance. Notably, suppression of FGFR4 dramatically diminishes glutathione synthesis and Fe2+ efflux efficiency via the β-catenin/TCF4-SLC7A11/FPN1 axis, resulting in excessive ROS production and labile iron pool accumulation. Ferroptosis, a unique iron-dependent form of oxidative cell death, is triggered after FGFR4 inhibition. Experiments involving patient-derived xenografts and organoids reveals a synergistic effect of anti-FGFR4 with anti-HER2 therapy in breast cancer with either intrinsic or acquired resistance. Together, these results pinpoint a mechanism of anti-HER2 resistance and provide a strategy for overcoming resistance via FGFR4 inhibition in recalcitrant HER2-positive breast cancer.

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