Secondary lymphedema is a debilitating condition driven by impaired regeneration of lymphatic vasculature following lymphatic injury, surgical removal of lymph nodes in cancer patients, or infection. However, the extent to which collecting lymphatic vessels regenerate following injury remains unclear. Here, we employed a novel mouse model of lymphatic injury in combination with state-of-the-art lymphatic imaging to demonstrate that the implantation of an optimized fibrin gel following lymphatic vessel injury leads to the reconnection of the injured lymphatic vessel network through sprouting lymphangiogenesis of initial-like lymphatic vessels from the ends of the collecting lymphatic vessels, resulting in the restoration of lymph flow to the draining lymph node. Mechanistically, we found that fibrin implantation elevates the tissue levels of CCL5, a potent immune cell-recruiting chemokine. Notably, injured vessels in CCL5-KO mice made fewer connections following fibrin gel implantation. These novel findings shed light on the mechanisms underlying lymphatic regeneration and suggest that enhancing CCL5 signaling may be a promising therapeutic strategy for enhancing lymphatic regeneration.

Supplementary Fig. S9: Expression levels of Cxcr3 and its ligands are increased in ICC tissues after GC/dual ICB treatment in murine 425-ICC.

Supplementary Fig. S8: Bulk tissue RNA sequencing analysis of ICC after GC/dual ICB combination therapy in orthotopic murine 425-ICC model.

Supplementary Fig. S12: Effect of ICB treatment scheduling on efficacy and toxicity.

Supplementary Table S1: Antibody list.

Supplementary Fig. S2: Standard chemotherapy converts ICB-resistant ICCs to ICB-responsive tumors, significantly delays tumor progression and increases survival in mice.

Supplementary Fig. S10: Schematic of the animal experiment design.

Supplementary Fig. S6: CTLA-4 blockade mediates the efficacy of GC/ICB therapy in ICC and increases CD8+CTL frequency in murine ICC.

Supplementary Fig. S11: Cxcr3 in CD8 T cells mediates the benefit of GC/ICB combination therapy in orthotopic murine 425-ICC model.

Supplementary Fig. S4: IMC analysis of ICC after GC-based therapies in orthotopic murine 425-ICC model.

Supplementary Fig. S7: Immunophenotyping in murine 425-ICC and SS49-ICC.

Supplementary Fig. S5: CTLA-4 blockade is critical for efficacy of combined chemotherapy with ICB in ICC by grafting 425 murine cells in C57Bl/6/FVB F1 mice.

Supplementary Fig. S3: ScRNAseq analysis of ICC TME after GC-based therapies in orthotopic murine 425-ICC model.

Supplementary Table S2: Primers for PCR.

Supplementary Fig. S1: Gating strategies for flow cytometry.

In recent decades, immunotherapy with chimeric antigen receptors (CARs) has revolutionized cancer treatment and given hope where other cancer therapies have failed. CAR-natural killer (NK) cells are NK cells that have been engineered ex vivo with a CAR on the cell membrane with high specificity for specific target antigens of tumor cells. The impressive results of several studies suggest that CAR-NK cell therapy has significant potential and successful performance in cancer treatment. Despite its effectiveness, CAR-NK cell therapy can have significant challenges when it comes to treating cancer. These challenges include tumor heterogeneity, antigen escape, an immunosuppressive tumor microenvironment, limited tissue migration from blood, exhaustion of CAR-NK cells, and inhibition by immunosuppressive checkpoint molecule signaling, etc. In CAR-T cell therapy, the use of combined approaches has shown encouraging outcomes for tumor regression and improved cancer treatment compared to single therapies. Therefore, to overcome these significant challenges in CAR-NK cells, innovative combination therapies of CAR-NK cells with other conventional therapies (e.g., chemotherapy and radiotherapy) or other immunotherapies are needed to counteract the above challenges and thereby increase the activity of CAR-NK cells. This review comprehensively discusses various cancer-treatment approaches in combination with CAR-NK cell therapy in the hope of providing valuable insights that may improve cancer treatment in the near future.

Purpose:Tumor hypoxia is a key barrier to successful delivery and activity of anti-cancer agents. To tackle this, we designed hypoxia-responsive Au-PEI-Azo-mPEG nanoparticles (NPs) denoted as APAP NPs for targeted delivery of hypoxia-activated prodrug (HAP), tirapazamine (TPZ) to hypoxic breast cancer cells. Methods:AuNPs were first synthesized. And then, were coated with polyethylene imine (PEI) by EDC-NHS chemistry. To realize NP biocompatibility and self-activating potential, a hypoxia-cleavable mPEG-AZO linker shell was coupled to the Au-PEI core. The hypoxia-responsible behavior of nanoparticles was analyzed under 21% O2 (normoxia) and 1% O2 (hypoxia) condition in 2D cell culture as well as MDA-MB-231 and MCF-7 spheroids as reliable biomimetics of tumor hypoxia. Results:APAP NPs elicited comparable cytotoxicity upon MDA-MB-231 cancer cells lowering TPZ IC50 to 7.46 µg/mL after 24 h. And were capable of enhanced ROS generation (P<0.001), and reduced mitochondrial membrane potential under hypoxia condition compared to the control (P<0.0001). Further, these NPs induced widespread apoptosis in both 2D and 3D cancer cell culture (P<0.0001), significantly reduced cell adhesion density (P<0.01), increased cell uptake by ~100 folds under hypoxia condition, and destroyed large MCF-7 spheroids by 72 h. Conclusion:Together, APAP@TPZ as biocompatible, and multi-stage activating platforms afford deepened penetration of HAP to hypoxic tumor core, where PEG detachment and TPZ bioreduction into its active form promote selective and effective eradication of hypoxic breast cancer microtumors.

Immune checkpoint blockade therapy, including anti-PD-1 antibody (αPD1), has shown limited success in glioblastoma (GBM), a lethal brain tumor with a median survival of under two years. The Wnt signaling pathway - known for its role in development - can limit treatment outcomes in several malignancies, including GBM. Moreover, it plays a critical role in GBM progression by promoting stemness and epithelial-mesenchymal transition. While the preclinical evidence indicates that Wnt signaling is linked to anti-PD-1 (αPD1) resistance in extracranial tumors, its role in GBM immune suppression and αPD1 resistance remains unknown. Here, we show that Wnt7b is upregulated in GBM patients and contributes to αPD1 therapy resistance in a clinically relevant, stemness-rich murine GBM syngeneic model (GSC005). We observed that elevated Wnt7b and β-catenin levels correlated with a poor αPD1 response. We hypothesized that inhibiting Wnt signaling could sensitize tumors to αPD1 therapy by reprogramming the TME from immunosuppressive to immunostimulatory and bolster anti-tumor immunity. We orthotopically implanted GSC005 into immunocompetent mice, then tracked tumor growth using ultrasound, and analyzed tumor and immune cell subpopulations by flow cytometry. Combining the porcupine inhibitor WNT974 with αPD1 improved median survival from 25 to 59 Days (p < 0.05), with 25% of mice achieving long-term survival. WNT974 with αPD1 increased DC3-like dendritic cells and reduced granulocytic myeloid-derived suppressor cells (gMDSCs). Moreover, this combination increased the Ki67+ CD8 T/Ki67+ Treg ratio, shifting the balance towards an anti-tumor immune response and upregulated Gzmb in CD8 T cells, enhancing their effector function. Interestingly, resistance to WNT974 + αPD1 was marked by elevated monocytic MDSCs, pERK/MAPK pathway activation, and modest T cell exhaustion. Our study provides compelling data and rationale to repurpose WNT974, reported to be safe in a Phase I trial in extracranial cancer patients, in combination with αPD1 therapy in GBM patients with elevated Wnt7b/β-catenin signaling.