The development and progression of colorectal cancer (CRC) is closely related to gut microbiome. Here we investigated the impact of lipopolysaccharide (LPS), one of the most prevalent products in the gut microbiome, on CRC immunotherapy. We found that LPS was abundant in orthotopic CRC tissue and was associated with low responses to anti-PD-L1 mAb therapy, and clearance of Gram-negative bacteria from the gut using polymyxin B (PmB), or blockade of Toll-like receptor 4 using TAK-242, would both relieve the immunosuppressive microenvironment and boost T-cell infiltration into the CRC tumor. Further, we designed an engineered LPS-targeting fusion protein and loaded its coding sequence into a lipid-protamine-DNA (LPD) nanoparticle system for selectively expression of LPS trap protein and blocking LPS inside the tumor, and this nano-trapping system significantly relieved the immunosuppressive microenvironment and boosted anti-PD-L1 mAb therapy against CRC tumor. This LPS trap system even attenuated CRC liver metastasis when applied, suggesting the importance of blocking LPS in the gut-liver axis. The strategy applied here may provide a useful new way for treating CRC as well as other epithelial cancers that interact with mucosa microbiome.

For most cancer types, only a minority of cancer patients respond to checkpoint inhibition therapy. T lymphocyte infiltration is critically important for checkpoint blockade immunotherapy. High expression of high mobility group protein A1 (HMGA1) is observed in rapidly proliferating neoplastic cells, and is reported to contribute to the immunosuppressive microenvironment in the tumor. Herein, whether the silencing of HMGA1 using a nanoparticle (NP) approach could promote T lymphocyte infiltration into the tumor, and sensitize tumors to checkpoint inhibitor therapy in several orthotopic murine cancer models, which has high levels of HMGA1 but little T lymphocyte infiltration, is investigated. Selectively silencing HMGA1 using a lipid-protamine-hyaluronic acid-siHMGA1 (LPH-siHMGA1) NP system greatly enhances the lymphocyte infiltration in the tumor. Furthermore, the combination of LPH-siHMGA1 and a locally expressed PD-L1 inhibitor system, a lipid-protamine-DNA NP loaded with plasmid encoding the PD-L1 trap fusion protein, significantly inhibits the tumor growth and prolonged survival. LPH-siHMGA1 also decreased the content of stem cells in the tumor. These findings highlight the potential of targeting HMGA1, especially using a nano approach, in the combination with cancer immunotherapy, and provide a strategy for broadening the application and enhancing the efficacy of checkpoint inhibitors.

To understand the influence of carboxylation on the interaction of carbon nanotubes with cells, the amount of pristine multi-walled carbon nanotubes (P-MWNTs) or carboxylated multi-walled carbon nanotubes (C-MWNTs) coated with Pluronic® F-108 that were accumulated by macrophages was measured by quantifying CNTs extracted from cells. Mouse RAW 264.7 macrophages and differentiated human THP-1 (dTHP-1) macrophages accumulated 80–100 times more C-MWNTs than P-MWNTs during a 24-hour exposure at 37 °C. The accumulation of C-MWNTs by RAW 264.7 cells was not lethal; however, phagocytosis was impaired as subsequent uptake of polystyrene beads was reduced after a 20-h exposure to C-MWNTs. The selective accumulation of C-MWNTs suggested that there might be receptors on macrophages that bind C-MWNTs. The binding of C-MWNTs to macrophages was measured as a function of concentration at 4 °C in the absence of serum to minimize the potential interference by serum proteins or temperature-dependent uptake processes. The result was that the cells bound 8.7 times more C-MWNTs than P-MWNTs, consistent with the selective accumulation of C-MWNTs at 37 °C. In addition, serum strongly antagonized the binding of C-MWTS to macrophages, suggesting that serum contained inhibitors of binding. Moreover, inhibitors of class A scavenger receptor (SR-As) reduced the binding of C-MWNTs by about 50%, suggesting that SR-As contribute to the binding and endocytosis of C-MWNTs in macrophages but that other receptors may also be involved. Altogether, the evidence supports the hypothesis that macrophages contain binding sites selective for C-MWNTs that facilitate the high accumulation of C-MWNTs compared to P-MWNTs.

Endothelial cells line all blood vessels, where they coordinate blood vessel formation and the blood-tissue barrier via regulation of cell-cell junctions. The nucleus also regulates endothelial cell behaviors, but it is unclear how the nucleus contributes to endothelial cell activities at the cell periphery. Here, we show that the nuclear-localized linker of the nucleoskeleton and cytoskeleton (LINC) complex protein SUN1 regulates vascular sprouting and endothelial cell-cell junction morphology and function. Loss of murine endothelial Sun1 impaired blood vessel formation and destabilized junctions, angiogenic sprouts formed but retracted in SUN1-depleted sprouts, and zebrafish vessels lacking Sun1b had aberrant junctions and defective cell-cell connections. At the cellular level, SUN1 stabilized endothelial cell-cell junctions, promoted junction function, and regulated contractility. Mechanistically, SUN1 depletion altered cell behaviors via the cytoskeleton without changing transcriptional profiles. Reduced peripheral microtubule density, fewer junction contacts, and increased catastrophes accompanied SUN1 loss, and microtubule depolymerization phenocopied effects on junctions. Depletion of GEF-H1, a microtubule-regulated Rho activator, or the LINC complex protein nesprin-1 rescued defective junctions of SUN1-depleted endothelial cells. Thus, endothelial SUN1 regulates peripheral cell-cell junctions from the nucleus via LINC complex-based microtubule interactions that affect peripheral microtubule dynamics and Rho-regulated contractility, and this long-range regulation is important for proper blood vessel sprouting and junction integrity.

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