PDPN positive CAFs contribute to HER2 positive breast cancer resistance to trastuzumab by inhibiting antibody-dependent NK cell-mediated cytotoxicity.

Trastuzumab is a humanized monoclonal antibody, and has been clinical employed to treat human epidermal growth factor receptor 2 (HER2) positive breast cancer. However, drug resistance to trastuzumab remains a challenge due to the generally uncharacterized interactive immune responses within the tumor tissue. In this study, by means of single-cell sequencing, we identified a novel podoplanin-positive (PDPN+) cancer-associated fibroblasts (CAFs) subset, which was enriched in trastuzumab resistant tumor tissues. Furthermore, we found that PDPN+ CAFs promote resistance to trastuzumab in HER2+ breast cancer by secreting immunosuppressive factors indoleamine 2,3-dioxygenase 1 (IDO1) as well as tryptophan 2,3-dioxygenase 2 (TDO2), thereby suppressing antibody-dependent cell-mediated cytotoxicity (ADCC), which was mediated by functional NK cells. A dual inhibitor IDO/TDO-IN-3 simultaneously targeting IDO1 and TDO2 showed a promising effect on reversing PDPN+ CAFs-induced suppression of NK cells mediated ADCC. Collectively, a novel subset of PDPN+ CAFs was identified in this study, which induced trastuzumab resistance in breast cancer of HER2+ status via inhibiting ADCC immune response mediated by NK cells, hinting that PDPN+ CAFs could be a novel target of treatment to increase the sensitivity of HER2+ breast cancer to trastuzumab.

Epitranscriptomic editing of the RNA N6-methyladenosine modification by dCasRx conjugated methyltransferase and demethylase.

N6-methyladenosine (m6A) is a common modification on endogenous RNA transcripts in mammalian cells. Technologies to precisely modify the RNA m6A levels at specific transcriptomic loci empower interrogation of biological functions of epitranscriptomic modifications. Here, we developed a bidirectional dCasRx epitranscriptome editing platform composed of a nuclear-localized dCasRx conjugated with either a methyltransferase, METTL3, or a demethylase, ALKBH5, to manipulate methylation events at targeted m6A sites. Leveraging this platform, we specifically and efficiently edited m6A modifications at targeted sites, reflected in gene expression and cell proliferation. We employed the dCasRx epitranscriptomic editor system to elucidate the molecular function of m6A-binding proteins YTHDF paralogs (YTHDF1, YTHDF2 and YTHDF3), revealing that YTHDFs promote m6A-mediated mRNA degradation. Collectively, our dCasRx epitranscriptome perturbation platform permits site-specific m6A editing for delineating of functional roles of individual m6A modifications in the mammalian epitranscriptome.

CD63+ cancer-associated fibroblasts confer CDK4/6 inhibitor resistance to breast cancer cells by exosomal miR-20.

Cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors (CDK4/6i) have rapidly received Food and Drug Administration (FDA) approval as a new type of therapy for patients with advanced hormone receptor-positive breast cancer. However, with the widespread application of CDK4/6i, drug resistance has become a new challenge for clinical practice and has greatly limited the treatment effect. Here, the whole microenvironment landscape of ER+ breast cancer tumors was revealed through single-cell RNA sequencing, and a specific subset of cancer-associated fibroblasts (CD63+ CAFs) was identified as highly enriched in CDK4/6i resistant tumor tissues. Then, we found that CD63+ CAFs can distinctly promote resistance to CDK4/6i in breast cancer cells and tumor xenografts. In addition, it was discovered that miR-20 is markedly enriched in the CD63+ CAFs-derived exosomes, which are used to communicate with ER+ breast cancer cells, leading to CDK4/6i resistance. Furthermore, exosomal miR-20 could directly target the RB1 mRNA 3'UTR and negatively regulate RB1 expression to decrease CDK4/6i sensitivity in breast cancer cells. Most importantly, we designed and synthesized cRGD-miR-20 sponge nanoparticles and found that they can enhance the therapeutic effect of CDK4/6i in breast cancer. In summary, our findings reveal that CD63+ CAFs can promote CDK4/6i resistance via exosomal miR-20, which induces the downregulation of RB1 in breast cancer cells, and suggest that CD63+ CAFs may be a novel therapeutic target to enhance CDK4/6i sensitivity.

Engineering CAR-NK cell derived exosome disguised nano-bombs for enhanced HER2 positive breast cancer brain metastasis therapy.

HER2-positive breast cancer brain metastasis (HER2+ BCBM) is a refractory malignancy with a high recurrence rate and poor prognosis. The efficacies of conventional treatments, including radiation and the FDA-approved drug trastuzumab, are compromised due to their significant obstacles, such as limited penetration through the blood-brain barrier (BBB), off-target effects on HER2+ tumor cells, and systemic adverse reactions, ultimately resulting in suboptimal therapeutic outcomes. In order to address these challenges, a novel biomimetic nanoplatform was created, which consisted of a combination of chimeric antigen receptor-natural killer (CAR-NK) cell-derived exosomes (ExoCAR), and a nanobomb (referred to as Micelle). This nanoplatform, known as ExoCAR/T7@Micelle, was designed to enhance the effectiveness of antitumor treatment by disrupting ferroptosis defense mechanisms. Due to the transferrin receptor binding peptide (T7) modification and CAR expression on the exosome surface, the nanoplatform successfully traversed the blood-brain barrier and selectively targeted HER2+ breast cancer cells. Moreover, integration of the reactive oxygen species (ROS) -amplified and photodynamic therapy (PDT)-based nanobomb facilitated the spatiotemporal release of the cargos at specific sites. Upon systemic administration of ExoCAR/T7@Micelle, mice with orthotopic HER2+ BCBM demonstrated a robust antitumor response in vivo, leading to a significant extension in survival time. Furthermore, histological analyses and blood index studies revealed no discernible side effects. Collectively, this study is the first to indicate the possibility of HER2+ BCBM therapy with a CAR-NK cell-derived biomimetic drug delivery system.