PA suppresses antitumor immunity of T cells by disturbing mitochondrial activity through Akt/mTOR-mediated Ca2+ flux.

Deciphering the mechanisms behind how T cells become exhausted and regulatory T cells (Tregs) differentiate in a tumor microenvironment (TME) will significantly benefit cancer immunotherapy. A common metabolic alteration feature in TME is lipid accumulation, associated with T cell exhaustion and Treg differentiation. However, the regulatory role of free fatty acids (FFA) on T cell antitumor immunity has yet to be clearly illustrated. Our study observed that palmitic acid (PA), the most abundant saturated FFA in mouse plasma, enhanced T cell exhaustion and Tregs population in TME and increased tumor growth. In contrast, oleic acid (OA), a monounsaturated FFA, rescued PA-induced T cell exhaustion, decreased Treg population, and ameliorated T cell antitumor immunity in an obese mouse model. Mechanistically, mitochondrial metabolic activity is critical in maintaining T cell function, which PA attenuated. PA-induced T cell exhaustion and Treg formation depended on CD36 and Akt/mTOR-mediated calcium signaling. The study described a new mechanism of PA-induced downregulation of antitumor immunity of T cells and the therapeutic potential behind its restoration by targeting PA.

Novel fusion protein PK5-RL-Gal-3C inhibits hepatocellular carcinoma via anti-angiogenesis and cytotoxicity.

BACKGROUND: Galectin-3 (Gal-3), the only chimeric ß-galactosides-binding lectin, consists of Gal-3N (N-terminal regulatory peptide) and Gal-3C (C-terminal carbohydrate-recognition domain). Interestingly, Gal-3C could specifically inhibit endogenous full-length Gal-3 to exhibit anti-tumor activity. Here, we aimed to further improve the anti-tumor activity of Gal-3C via developing novel fusion proteins. METHODS: PK5 (the fifth kringle domain of plasminogen) was introduced to the N-terminus of Gal-3C via rigid linker (RL) to generate novel fusion protein PK5-RL-Gal-3C. Then, we investigated the anti-tumor activity of PK5-RL-Gal-3C in vivo and in vitro by using several experiments, and figured out their molecular mechanisms in anti-angiogenesis and cytotoxicity to hepatocellular carcinoma (HCC). RESULTS: Our results show that PK5-RL-Gal-3C can inhibit HCC both in vivo and in vitro without obvious toxicity, and also significantly prolong the survival time of tumor-bearing mice. Mechanically, we find that PK5-RL-Gal-3C inhibits angiogenesis and show cytotoxicity to HCC. In detail, HUVEC-related and matrigel plug assays indicate that PK5-RL-Gal-3C plays an important role in inhibiting angiogenesis by regulating HIF1α/VEGF and Ang-2 both in vivo and in vitro. Moreover, PK5-RL-Gal-3C induces cell cycle arrest at G1 phase and apoptosis with inhibition of Cyclin D1, Cyclin D3, CDK4, and Bcl-2, but activation of p27, p21, caspase-3, -8 and -9. CONCLUSION: Novel fusion protein PK5-RL-Gal-3C is potent therapeutic agent by inhibiting tumor angiogenesis in HCC and potential antagonist of Gal-3, which provides new strategy for exploring novel antagonist of Gal-3 and promotes their application in clinical treatment.

Herpes Virus Entry Mediator Costimulation Signaling Enhances CAR T-cell Efficacy Against Solid Tumors Through Metabolic Reprogramming.

Costimulatory domains (CSD) of 4-1BB and CD28 are most widely used in chimeric antigen receptor (CAR)-engineered T cells. These CAR T cells have shown encouraging efficacy in the treatment of hematologic malignancies but have limited efficacy in solid tumors. The herpes virus entry mediator (HVEM) is a costimulatory molecule with a novel downstream signaling pathway. In response to target cells, CAR T cells with a HVEM CSD (HVEM-CAR T) displayed more robust cytokine release and cytotoxicity than 4-1BB-CAR T or CD28-CAR T in vitro. Furthermore, HVEM-CAR T showed superior therapeutic efficacy in several mouse tumor models. Mechanistically, the HVEM CSD endowed CAR T cells with attenuated exhaustion, improved function and persistence, and enhanced metabolic activities in tumor tissue compared with 4-1BB-based or CD28-based CAR T cells. These studies establish that the HVEM CSD has the potential to improve the therapeutic efficacy of CAR T cells against solid tumors.

Roux-en-Y reconstruction alleviates radical gastrectomy-induced colitis via down-regulation of the butyrate/NLRP3 signaling pathway.

BACKGROUND: Different methods for digestive tract reconstruction have a complex impact on the nutritional status of gastric cancer (GC) patients after radical gastrectomy. Previous studies reported that Roux-en-Y (R-Y) reconstruction resulted in obvious weight reduction and improvement in type 2 diabetes in obese patients. We investigated the relationship between R-Y reconstruction, gut microbiota, and the NLRP3 inflammasome in GC patients with poor basic nutrition. METHODS: Changes in the gut microbiota after radical gastrectomy accomplished by different methods of digestive tract reconstruction were investigated via fecal microbiota transplantation. The underlying mechanisms were also explored by analyzing the role of the microbiota, butyrate, and the NLRP3 inflammasome in the colon tissues of colitis model mice and GC patients after radical gastrectomy. FINDINGS: R-Y reconstruction effectively relieved intestinal inflammation and facilitated nutrient absorption. 16S rRNA analysis revealed that gavage transplantation with the fecal microbiota of R-Y reconstruction patients could reverse dysbacteriosis triggered by radical gastrectomy and elevate the relative abundance of some short-chain fatty acid (SCFA)-producing bacteria. Subsequently, butyrate negatively regulated the NLRP3-mediated inflammatory signaling pathway to inhibit the activation of macrophages and the secretion of pro-inflammatory mediators such as caspase-1 and interleukin (IL)-1ß, decreasing the level of intestinal inflammation and promoting nutrient absorption. INTERPRETATION: R-Y reconstruction induced colonization with SCFA-producing bacteria to alleviate radical gastrectomy-induced colitis by down-regulating the NLRP3 signaling pathway. This can be a new strategy and theoretical basis for the management of the postoperative nutritional status of GC patients. FUNDING: This work was supported by the National Nature Science Foundation of China (81974375), the BoXi cultivation program (BXQN202130), and the Project of Youth Foundation in Science and Education of the Department of Public Health of Suzhou (KJXW2018001).

Docetaxel enhances the therapeutic efficacy of PSMA-specific CAR-T cells against prostate cancer models by suppressing MDSCs.

PURPOSE: Prostate cancer can undergo curative effects by radical prostatectomy or radical radiotherapy. However, the best treatment for more aggressive high-risk prostate cancer remains controversial. Insufficient infiltration capacity and dysfunction are commonly occurrences in engineered T lymphocytes expressing chimeric antigen receptor (CAR-T), characterizing cancer immunotherapy failure. We conducted this study to investigate whether the combinative application of docetaxel and PSMA-CAR-T cells could be a more effective treatment to prostate cancer. METHODS: Expressions of prostate specific membrane antigen (PSMA) on prostate cancer cells were examined by Flow cytometry. The efficaciousness of PSMA-CAR-T was evaluated in vitro using ELISA and RTCA. The effect of intermixed therapy was assessed in vivo utilizing a human prostate cancer liver metastasis mouse model and a human prostate cancer cell xenograft mouse model. RESULTS: The outcome of cytokine discharge and cell killing assays demonstrated that PSMA-CAR-T cells have characteristic effector capacity against PSMA+ prostate cancer cells in vitro. Additionally, collaborative treatment of PSMA-CAR-T cells and docetaxel have cooperative efficacy in a mouse model of human prostate cancer. The merged strategy could be seen as an undeveloped avenue to augmenting adoptive CAR-T cell immunotherapy and mitigating the adverse side effects of chemotherapy. CONCLUSIONS: Cooperation of PSMA-specific CAR-T cells and the chemotherapy drug docetaxel can impressively ameliorate antitumor effectiveness against an installed metastatic human prostate cancer model in NPG mice.

CD36 regulates LPS-induced acute lung injury by promoting macrophages M1 polarization.

M1 polarization of macrophages works as a promoter in pathogenesis of acute lung injury / acute respiratory distress syndrome (ALI/ARDS) by the secretion of pro-inflammatory cytokines and recruiting other inflammatory cells. Lipopolysaccharide (LPS), a critical component of the wall of gram-negative bacteria, can induce M1 polarization and ALI. Recently, cluster of differentiation 36 (CD36) has been reported to be associated with inflammatory responses. However, it has not yet been clarified whether CD36 in macrophages is involved in LPS-induced ALI. Herein, we demonstrated that in macrophages, LPS-induced ALI was regulated by CD36. Loss of CD36 attenuated LPS-induced ALI by reducing M1 polarization. Mechanistically, CD36 promoted macrophage M1 polarization by regulating CD14 associated with TLR4 during LPS stimulation. The findings of this study, clarified the mechanism of LPS-induced ALI through CD36 in macrophages, which provides a potential target for the prevention and treatment of ALI.

Bacteroides fragilis restricts colitis-associated cancer via negative regulation of the NLRP3 axis.

Patients with persistent ulcerative colitis (UC) are at a higher risk of developing colitis-associated cancer (CAC). Previous studies have reported that intestinal microbiota disturbance plays an important role in the process of CAC development in patients with UC, indicating that targeted intervention of intestinal microbiota and its metabolites may be a potential therapeutic strategy. Gut microbiota in the process of colorectal cancer development in UC patients was analyzed using the gutMEGA database and verified in fecal samples. The abundance of Bacteroides fragilis reduced significantly in the process of colitis associated cancer development. Broad-spectrum antibiotics (BSAB) intervene with the intestinal microbiota of mice and accelerate the process of colon cancer development. However, gavage transplantation with B. fragilis can effectively reverse the effects of BSAB. In the intestinal tract, B. fragilis promotes the secretion of short-chain fatty acids (SCFAs). Subsequently, SCFAs, especially butyrate, negatively regulate the inflammatory signaling pathway mediated by NLRP3 to inhibit the activation of macrophages and the secretion of proinflammatory mediators such as IL-18 and IL-1ß, reducing the level of intestinal inflammation and restricting CAC development. In conclusion, colonization with B. fragilis has been shown to be effective in ameliorating intestinal epithelial damage caused by chronic inflammation and preventing the development of colonic tumors. Thus, it can be a therapeutic intervention strategy with good clinical application prospects.

Lenvatinib enhances T cell immunity and the efficacy of adoptive chimeric antigen receptor-modified T cells by decreasing myeloid-derived suppressor cells in cancer.

BACKGROUND: Lenvatinib, a tyrosine kinase inhibitor, has been approved for the treatment of several cancers. However, its regulatory activity and related mechanisms on T cell antitumour immunity need to be further investigated. METHODS: The antitumour activity of lenvatinib in immunocompetent and immunodeficient mice was compared to determine the role of T cell immunity. The antitumour activity of T cells was analysed by cytokine production and adoptive T cell therapy. The immunosuppressive effects of MDSCs on T cells were determined by detecting cytokine production in T cells after being cocultured with MDSCs. The adjuvant immunotherapy effect of lenvatinib was determined by combination therapy with CAR-T cells targeted carbonic anhydrase IX (CAIX) in a murine renal cancer model. RESULTS: The antitumour activity of lenvatinib was greater in immunocompetent mice than in immunodeficient mice and was attenuated by CD8+T cell depletion. Lenvatinib increased proliferation, tumour infiltration and antitumour activity of T cells. Importantly, adoptive transfer of lenvatinib-treated T cells showed a long-term antitumour response in vivo. Mechanistically, lenvatinib upregulated T cell-related chemokines (CXCL10 and CCL8) in tumours and decreased the frequency and immunosuppressive activity of MDSCs. Furthermore, lenvatinib enhanced the efficacy of CAR-T cells in a murine renal cancer model. CONCLUSION: Our study revealed novel antitumour mechanisms of lenvatinib by enhancing T cell-mediated antitumour immunity. These findings are of great significance for guiding the clinical use of lenvatinib and provide a good candidate for future combination therapy with T-cell therapies or other immunotherapies.

Tyrosine Kinase Inhibitor Cabozantinib Inhibits Murine Renal Cancer by Activating Innate and Adaptive Immunity.

BACKGROUND: Advanced renal cell carcinoma (RCC) has a very dismal prognosis. Cabozantinib, a tyrosine kinase inhibitor, has been approved for the treatment of advanced RCC. However, the impact of cabozantinib on the immune microenvironment of RCC remains poorly understood. METHODS: Kaplan-Meier survival curves were constructed to examine the correlation between intratumor infiltration of neutrophils and patient prognosis in RCC. Infiltration and effector function of neutrophils and T cells in response to cabozantinib treatment were investigated in a murine RCC model. RESULTS: A retrospective study of 307 RCC patients indicated that neutrophils were recruited into tumor tissues, and increased neutrophil infiltration was associated with improved clinical outcomes. In a murine model of RCC, cabozantinib treatment significantly increased both intratumor infiltration and anti-tumor function of neutrophils and T cells. Mechanistically, we found that cabozantinib treatment induced expression of neutrophil-related chemokines (CCL11 and CXCL12) and T cell-related chemokines (CCL8 and CX3CL1) in the tumor microenvironment. Furthermore, depletion of neutrophils and CD8+ T cells compromised the therapeutic efficacy of cabozantinib. Importantly, cabozantinib treatment induced long-term anti-tumor T cell response. CONCLUSIONS: Our study revealed novel mechanisms of the therapeutic effects of cabozantinib on RCC by activating both neutrophil-mediated innate immunity and T cell-mediated adaptive immunity. These findings are of great significance for guiding the clinical use of cabozantinib and provide a good candidate for future combination therapy with T-cell therapies or other immunotherapies.

Akt inhibition at the initial stage of CAR-T preparation enhances the CAR-positive expression rate, memory phenotype and in vivo efficacy.

The adoptive transfer of chimeric antigen receptor-modified T (CAR-T) cells is a novel cancer treatment that has led to encouraging breakthroughs in the treatment of haematological malignancies. The efficacy of infused CAR-T cells is associated with a high CAR-positive expression rate, a strong proliferative response and the persistence of CAR-T cells in vivo. Manufacturing CAR-T cells is a process usually associated with the decreased CAR-positive expression rate and terminal differentiation of the infused CAR-T cells, which causes decreased proliferation and persistence of CAR-T cells in vivo. Therefore, the preparation of a high CAR-positive expression rate and few differentiated CAR-T cells is particularly important for clinical cancer treatment. In this study, we transduced and expanded CAR-T cells targeting the epithelial cell adhesion molecule (EpCAM) in the presence of an Akt inhibitor (MK2206) during the initial stage of CAR-T cell preparation. We show that the Akt inhibitor did not suppress the proliferation or effector function of the EpCAM-CAR-T cells but increased the CAR-positive expression rate and decreased the number of terminally differentiated EpCAM-CAR-T cells. Furthermore, EpCAM-CAR-T cells prepared using this protocol appeared to have enhanced antitumor activity in vivo. Taken together, these findings suggest that Akt inhibition during the initial stage of CAR-T cell preparation could improve the performance of CAR-T cells.

Lenvatinib promotes antitumor immunity by enhancing the tumor infiltration and activation of NK cells.

Based on previous reports, the efficacy of lenvatinib against cancer is mainly attributed to its antiangiogenic activity and its ability to suppress tumor proliferation, which are mediated by targeting receptor tyrosine kinases (RTKs). However, the effects of lenvatinib on tumor immune modulation have rarely been explored. Here, we show that lenvatinib effectively inhibited murine melanoma and renal cancer, and this inhibition was associated with enhanced tumor infiltration by natural killer (NK) cells. Critically, lenvatinib-induced tumor growth inhibition was attenuated by antibody-mediated NK cell depletion or the blockade of NK cell chemotaxis with an anti-CXCR3 blocking antibody. In addition, the expression of natural cytotoxicity receptors (NCRs) by tumor-infiltrating NK cells and the expression of cytotoxic cytokines in the tumor tissue were also augmented by lenvatinib. These data thus suggest that lenvatinib may be used not only as a direct cytotoxic drug against tumor angiogenesis and proliferation but also as a potent adjunct for enhancing the efficacy of immune-based cancer therapies by enhancing the tumor infiltration and activation of NK cells.

Fatty acid 2-hydroxylation inhibits tumor growth and increases sensitivity to cisplatin in gastric cancer.

BACKGROUND: Most gastric cancers are diagnosed at an advanced or metastatic stage with poor prognosis and survival rate. Fatty acid 2-hydroxylase (FA2H) with high expression in stomach generates chiral (R)-2-hydroxy FAs ((R)-2-OHFAs) and regulates glucose utilization which is important for cell proliferation and invasiveness. We hypothesized that FA2H impacts gastric tumor growth and could represent a novel target to improve gastric cancer therapy. METHODS: FA2H level in 117 human gastric tumors and its association with tumor growth, metastasis and overall survival were examined. Its roles and potential mechanisms in regulating tumor growth were studied by genetic and pharmacological manipulation of gastric cancer cells in vitro and in vivo. FINDINGS: FA2H level was lower in gastric tumor tissues as compared to surrounding tissues and associated with clinicopathologic status of patients, which were confirmed by analyses of multiple published datasets. FA2H depletion decreased tumor chemosensitivity, partially due to inhibition of AMPK and activation of the mTOR/S6K1/Gli1 pathway. Conversely, FA2H overexpression or treatment with (R)-2-OHFAs had the opposite effects. In line with these in vitro observations, FA2H knockdown promoted tumor growth with increased level of tumor Gli1 in vivo. Moreover, (R)-2-OHFA treatment significantly decreased Gli1 level in gastric tumors and enhanced tumor chemosensitivity to cisplatin, while alleviating the chemotherapy-induced weight loss in mice. INTERPRETATION: Our results demonstrate that FA2H plays an important role in regulating Hh signaling and gastric tumor growth and suggest that (R)-2-OHFAs could be effective as nontoxic wide-spectrum drugs to promote chemosensitivity. FUND: Grants of NSF, NIH, and PAPD.

Ubiquitinated CD36 sustains insulin-stimulated Akt activation by stabilizing insulin receptor substrate 1 in myotubes.

Both the magnitude and duration of insulin signaling are important in executing its cellular functions. Insulin-induced degradation of insulin receptor substrate 1 (IRS1) represents a key negative feedback loop that restricts insulin signaling. Moreover, high concentrations of fatty acids (FAs) and glucose involved in the etiology of obesity-associated insulin resistance also contribute to the regulation of IRS1 degradation. The scavenger receptor CD36 binds many lipid ligands, and its contribution to insulin resistance has been extensively studied, but the exact regulation of insulin sensitivity by CD36 is highly controversial. Herein, we found that CD36 knockdown in C2C12 myotubes accelerated insulin-stimulated Akt activation, but the activated signaling was sustained for a much shorter period of time as compared with WT cells, leading to exacerbated insulin-induced insulin resistance. This was likely due to enhanced insulin-induced IRS1 degradation after CD36 knockdown. Overexpression of WT CD36, but not a ubiquitination-defective CD36 mutant, delayed IRS1 degradation. We also found that CD36 functioned through ubiquitination-dependent binding to IRS1 and inhibiting its interaction with cullin 7, a key component of the multisubunit cullin-RING E3 ubiquitin ligase complex. Moreover, dissociation of the Src family kinase Fyn from CD36 by free FAs or Fyn knockdown/inhibition accelerated insulin-induced IRS1 degradation, likely due to disrupted IRS1 interaction with CD36 and thus enhanced binding to cullin 7. In summary, we identified a CD36-dependent FA-sensing pathway that plays an important role in negative feedback regulation of insulin activation and may open up strategies for preventing or managing type 2 diabetes mellitus.

Major role of adipocyte prostaglandin E2 in lipolysis-induced macrophage recruitment.

Obesity induces accumulation of adipose tissue macrophages (ATMs), which contribute to both local and systemic inflammation and modulate insulin sensitivity. Adipocyte lipolysis during fasting and weight loss also leads to ATM accumulation, but without proinflammatory activation suggesting distinct mechanisms of ATM recruitment. We examined the possibility that specific lipid mediators with anti-inflammatory properties are released from adipocytes undergoing lipolysis to induce macrophage migration. In the present study, we showed that conditioned medium (CM) from adipocytes treated with forskolin to stimulate lipolysis can induce migration of RAW 264.7 macrophages. In addition to FFAs, lipolytic stimulation increased release of prostaglandin E2(PGE2) and prostaglandin D2(PGD2), reflecting cytosolic phospholipase A2α activation and enhanced cyclooxygenase (COX) 2 expression. Reconstituted medium with the anti-inflammatory PGE2potently induced macrophage migration while different FFAs and PGD2had modest effects. The ability of CM to induce macrophage migration was abolished by treating adipocytes with the COX2 inhibitor sc236 or by treating macrophages with the prostaglandin E receptor 4 antagonist AH23848. In fasted mice, macrophage accumulation in adipose tissue coincided with increases of PGE2levels and COX1 expression. Collectively, our data show that adipocyte-originated PGE2with inflammation suppressive properties plays a significant role in mediating ATM accumulation during lipolysis.