Renal Clearable Nanodots-Engineered Erythrocytes with Enhanced Circulation and Tumor Accumulation for Photothermal Therapy of Hepatocellular Carcinoma.

Living cell-mediated nanodelivery system is considered a promising candidate for targeted antitumor therapy; however, their use is restricted by the adverse interactions between carrier cells and nanocargos. Herein, a novel erythrocyte-based nanodelivery system is developed by assembling renal-clearable copper sulfide (CuS) nanodots on the outer membranes of erythrocytes via a lipid fusion approach, and demonstrate that it is an efficient photothermal platform against hepatocellular carcinoma. After intravenous injection of the nanodelivery system, CuS nanodots assembled on erythrocytes can be released from the system, accumulate in tumors in response to the high shear stress of bloodstream, and show excellent photothermal antitumor effect under the near infrared laser irradiation. Therefore, the erythrocyte-mediated nanodelivery system holds many advantages including prolonged blood circulation duration and enhanced tumor accumulation. Significantly, the elimination half-life of the nanodelivery system is 74.75 ± 8.77 h, which is much longer than that of nanodots (33.56 ± 2.36 h). Moreover, the other two kinds of nanodots can be well assembled onto erythrocytes to produce other erythrocyte-based hitchhiking platforms. Together, the findings promote not only the development of novel erythrocyte-based nanodelivery systems as potential platforms for tumor treatment but also their further clinical translation toward personalized healthcare.

A novel human single-domain antibody-drug conjugate targeting CEACAM5 exhibits potent in vitro and in vivo antitumor activity.

Leveraging the specificity of antibody to deliver cytotoxic agent into tumor, antibody-drug conjugates (ADCs) have become one of the hotspots in the development of anticancer therapies. Although significant progress has been achieved, there remain challenges to overcome, including limited penetration into solid tumors and potential immunogenicity. Fully human single-domain antibodies (UdAbs), with their small size and human nature, represent a promising approach for addressing these challenges. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is a glycosylated cell surface protein that rarely expressed in normal adult tissues but overexpressed in diverse cancers, taking part in tumorigenesis, progression, and metastasis. In this study, we investigated the therapeutic potential of UdADC targeting CEACAM5. We performed biopanning in our library and obtained an antibody candidate B9, which bound potently and specifically to CEACAM5 protein (KD = 4.84 nM) and possessed excellent biophysical properties (low aggregation tendency, high homogeneity, and thermal stability). The conjugation of B9 with a potent cytotoxic agent, monomethyl auristatin E (MMAE), exhibited superior antitumor efficacy against CEACAM5-expressing human gastric cancer cell line MKN-45, human pancreatic carcinoma cell line BxPC-3 and human colorectal cancer cell line LS174T with IC50 values of 38.14, 25.60, and 101.4 nM, respectively. In BxPC-3 and MKN-45 xenograft mice, administration of UdADC B9-MMAE (5 mg/kg, i.v.) every 2 days for 4 times markedly inhibited the tumor growth without significant change in body weight. This study may have significant implications for the design of next-generation ADCs.

A highly stable human single-domain antibody-drug conjugate exhibits superior penetration and treatment of solid tumors.

The inefficient tumor penetration of therapeutic antibodies has hampered their effective use in treating solid tumors. Here, we report the identification of a fully human single-domain antibody (UdAb), designated as n501, targeting the oncofetal antigen 5T4. The high-resolution crystal structure indicates that n501 adopts a compact structure very similar to that of camelid nanobodies, and binds tightly to all eight leucine-rich repeats of 5T4. Furthermore, the UdAb n501 exhibits exceptionally high stability, with no apparent activity changes over 4 weeks of storage at various temperatures. Importantly, the UdAb-based antibody-drug conjugate (n501-SN38) showed much deeper tumor penetration, significantly higher tumor uptake, and faster accumulation at tumor sites than conventional IgG1-based antibody-drug conjugate (m603-SN38), resulting in improved tumor inhibition. These results highlight the potential of UdAb-based antibody-drug conjugates as a potential class of antitumor therapeutics with characteristics of high stability and strong tumor penetration for the effective treatment of solid tumors.

Constitutive ERK1/2 activation contributes to production of double minute chromosomes in tumour cells.

Double minute chromosomes (DMs) are extrachromosomal cytogenetic structures found in tumour cells. As hallmarks of gene amplification, DMs often carry oncogenes and drug-resistance genes and play important roles in malignant tumour progression and drug resistance. The mitogen-activated protein kinase (MAPK) signalling pathway is frequently dysregulated in human malignant tumours, which induces genomic instability, but it remains unclear whether a close relationship exists between MAPK signalling and DMs. In the present study, we focused on three major components of MAPK signalling, ERK1/2, JNK1/2/3 and p38, to investigate the relationship between MAPK and DM production in tumour cells. We found that the constitutive phosphorylation of ERK1/2, but not JNK1/2/3 and p38, was closely associated with DMs in tumour cells. Inhibition of ERK1/2 activation in DM-containing and ERK1/2 constitutively phosphorylated tumour cells was able to markedly decrease the number of DMs, as well as the degree of amplification and expression of DM-carried genes. The mechanism was found to be an increasing tendency of DM DNA to break, become enveloped into micronuclei (MNs) and excluded from the tumour cells during the S/G2 phases of the cell cycle, events that accompanied the reversion of malignant behaviour. Our study reveals a linkage between ERK1/2 activation and DM stability in tumour cells.

Half-life extension of single-domain antibody-drug conjugates by albumin binding moiety enhances antitumor efficacy.

Single-domain antibody-drug conjugates (sdADCs) have been proven to have deeper solid tumor penetration and intratumor accumulation capabilities due to their smaller size compared with traditional IgG format ADCs. However, one of the key challenges for improving clinical outcomes of sdADCs is their abbreviated in vivo half-life. In this study, we innovatively fused an antihuman serum albumin (αHSA) nanobody to a sdADCs targeting oncofetal antigen 5T4, conferring serum albumin binding to enhance the pharmacokinetic profiles of sdADCs. The fusion protein was conjugated with monomethyl auristatin E (MMAE) at s224c site mutation. The conjugate exhibited potent cytotoxicity against various tumor cells. Compared with the nonalbumin-binding counterparts, the conjugate exhibited a 10-fold extended half-life in wild-type mice and fivefold prolonged serum half-life in BxPC-3 xenograft tumor models as well as enhanced tumor accumulation and retention in mice. Consequently, n501-αHSA-MMAE showed potent antitumor effects, which were comparable to n501-MMAE in pancreatic cancer BxPC-3 xenograft tumor models; however, in human ovarian teratoma PA-1 xenograft tumor models, n501-αHSA-MMAE significantly improved antitumor efficacy. Moreover, the conjugate showed mitigated hepatotoxicity. In summary, our results suggested that fusion to albumin-binding moiety as a viable strategy can enhance the therapeutic potential of sdADCs through optimized pharmacokinetics.

Cultivated Artemisia rupestris L. polysaccharide CARP2 as an adjuvant for influenza vaccines to prolong immune responses.

In the study, the adjuvant features of the immunoregulatory polysaccharide component CARP2 isolated from cultivated Artemisia rupestris L. for influenza virus vaccine (IVV) and the mechanism responsible for its action in DCs were further explored. CARP2 showed a typical absorbance peak of polysaccharides in spectral analysis. At two doses of CARP2-adjuvanted IVV, IgG, hemagglutination inhibition (HI) titers, and effector/memory T cells were generated and lasted for 275 days without adverse events. CARP2 primed rapid HI and IgG, IgG2a/IgG1 ratio, splenocyte proliferation, and cytotoxic T lymphocyte (CTL), and facilitated the generation of INF-γ and IL-4 by activating DCs and regulatory T cells (Tregs). Additionally, CARP2 achieved the ten-fold dose-sparing effect. In vitro, CARP2 stimulated DCs to prime the production of Th1/Th2 cytokines and CCR7 and activated MyD88-dependent pathway by upregulating the expressions of TLR4, MyD88, TRAF-6, and p65. In contrast, MyD88, TRAF-6, and NF-κB inhibitors partially blocked the effect through reducing related cytokines and proteins. Overall, CARP2 promoted IVV efficacy, which was involved in the modulation of Th1/Th2 responses and shifted toward Th1-polarizing response via TLR4/MyD88/TRAF/NF-κB activation in DCs.

Antibody-based cancer immunotherapy by targeting regulatory T cells.

Regulatory T cells (Tregs) are among the most abundant suppressive cells, which infiltrate and accumulate in the tumor microenvironment, leading to tumor escape by inducing anergy and immunosuppression. Their presence has been correlated with tumor progression, invasiveness and metastasis. Targeting tumor-associated Tregs is an effective addition to current immunotherapy approaches, but it may also trigger autoimmune diseases. The major limitation of current therapies targeting Tregs in the tumor microenvironment is the lack of selective targets. Tumor-infiltrating Tregs express high levels of cell surface molecules associated with T-cell activation, such as CTLA4, PD-1, LAG3, TIGIT, ICOS, and TNF receptor superfamily members including 4-1BB, OX40, and GITR. Targeting these molecules often attribute to concurrent depletion of antitumor effector T-cell populations. Therefore, novel approaches need to improve the specificity of targeting Tregs in the tumor microenvironment without affecting peripheral Tregs and effector T cells. In this review, we discuss the immunosuppressive mechanisms of tumor-infiltrating Tregs and the status of antibody-based immunotherapies targeting Tregs.

Immunoregulatory properties of the cultivated Artemisia rupestris L. polysaccharide as a potential adjuvant.

Two components (CARP-1 and CARP-2) were fractionated from cultivated Artemisia rupestris L. and then characterized by HPGPC and HPLC. CARP-1 with a molecular weight of 2.72 × 104 Da and CARP-2 with a molecular weight of 2.08 × 104 Da were mainly composed of galactose, arabinose, glucose and rhamnose. Polysaccharides were the active components as confirmed by the increased CD40, CD86, TNF-α, and IL-6, allogeneic T-cell activation, and reduced endocytosis in vitro assays. CARP-1 and CARP-2 at 10 to 3200 µg/mL was not cytotoxic to the splenocytes of mice. After immunization, CARP-1 and CARP-2 combined with OVA elicited mixed Th1/Th2 responses, especially polarized Th1 response. Furthermore, TLR4 inhibitor decreased CARP-1- and CARP-2-induced DC activation. Western blot revealed that CARP-1 and CARP-2 stimulated the phosphorylation changes of target proteins in NF-κB and MAPK pathways in a dose- or time-related manner. Overall, CARP-1 and CARP-2 could be exploited as an effective and safe adjuvant for vaccines.

Improving adjuvanticity of crude polysaccharides from cultivated Artemisia rupestris L. for influenza vaccine by promoting long-term immunity and TH1/TH2 response with dose-sparing effect.

ETHNOPHARMACOLOGICAL RELEVANCE: Influenza virus vaccines (IVV) with balanced TH1/TH2 responses are critical for controlling seasonal influenza. Emerging evidences suggest that herbal polysaccharides can induce potent TH1 or mixed TH1/TH2 responses. AIM OF STUDY: The study aims to determine the efficacy and safety of crude polysaccharides from cultivated Artemisia rupestris L. (CPCAR) as an adjuvant for IVV. MATERIALS AND METHODS: CPCAR was prepared with hot extraction and ethanol precipitation method and primary physico-chemical characters were tested. Mice were vaccinated by subcutaneous route with IVV formulated with different dose of CPCAR to detecting the elicited TH1/TH2 responses and long-term immune responses with dose-sparing sparing effect. RESULTS: IVV formulated with CPCAR without LPS contamination could augment balanced TH1/TH2 responses, as indicated by early IgG response, hemagglutination inhibition (HAI) antibodies, effector T-cells, and cytotoxic T lymphocytes (CTL). Moreover, CPCAR elicited long-term IgG, HAI antibodies, memory T cells, and balanced CD4/CD8 responses within 168 days after vaccination. Compared with IVV alone, a low or high dose of IVV formulated with CPCAR improved the levels of IgG, IgG1, and IgG2a and enhanced memory T cells and balanced CD4/CD8 responses, displaying a 10-fold dose-sparing effect. As determined by IgE response and monitoring results of weekly body weight and daily symptoms after vaccination, anaphylaxis or adverse effect was not observed. CONCLUSIONS: Collectively, the study demonstrated the potential of CPCAR as an aqueous polysaccharide adjuvant for IVV to induce rapid and balanced TH1/TH2 responses and long-lasting immunity with dose-sparing effect.

An antigen-strengthened dye-modified fully-human-nanobody-based immunoprobe for second near infrared bioimaging of metastatic tumors.

Conventional immunoprobes have absorption capabilities across the visible to near infrared (NIR-I, 650-900 nm) region. Recently, second near infrared (NIR-II, 1000-1700 nm) window have gained much attention due to their deeper penetration depth and improved visualization. Here, we describe the design and synthesis of a fully human nanobody-based fluorescent immunoprobe (ICGM-n501) for NIR-II bioimaging with strengthened fluorescent emission by antigen for the first time. By site-directed conjugation of an FDA-approved dye analogue, indocyanine green decorated with maleimide (ICGM), into a tumor-specific n501, ICGM-n501 provides real-time monitoring of abdominal transportation pathway of antibody-based bioagents with high resolution (0.21 mm), presents better accuracy and lower dosage (0.21 µmol kg-1) in bioimaging of peritoneal metastatic tumors than bioluminescence agent D-luciferin. In this work, ICGM-n501 demonstrates its potential in clinical surgery guidance, provide an expanded category of available NIR-II fluorophores and a template for next-generation immunoassay bioagents.

Novel Hypoxia-Related Gene Signature for Risk Stratification and Prognosis in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer with limited therapeutic options and low survival rate. The hypoxic microenvironment plays a vital role in progression, metabolism, and prognosis of malignancies. Therefore, this study aims to develop and validate a hypoxia gene signature for risk stratification and prognosis prediction of HCC patients. The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) databases were used as a training cohort, and one Gene Expression Omnibus database (GSE14520) was served as an external validation cohort. Our results showed that eight hypoxia-related genes (HRGs) were identified by the least absolute shrinkage and selection operator analysis to develop the hypoxia gene signature and demarcated HCC patients into the high- and low-risk groups. In TCGA, ICGC, and GSE14520 datasets, patients in the high-risk group had worse overall survival outcomes than those in the low-risk group (all log-rank P < 0.001). Besides, the risk score derived from the hypoxia gene signature could serve as an independent prognostic factor for HCC patients in the three independent datasets. Finally, a nomogram including the gene signature and tumor-node-metastasis stage was constructed to serve clinical practice. In the present study, a novel hypoxia signature risk model could reflect individual risk classification and provide therapeutic targets for patients with HCC. The prognostic nomogram may help predict individualized survival.

Gut Microbiota: Its Potential Roles in Pancreatic Cancer.

Pancreatic cancer is considered a lethal disease with a low survival rate due to its late-stage diagnosis, few opportunities for resection and lack of effective therapeutic strategies. Multiple, highly complex effects of gut microbiota on pancreatic cancer have been recognized as potential strategies for targeting tumorigenesis, development and treatment in recent decades; some of the treatments include antibiotics, probiotics, and fecal microbiota transplantation. Several bacterial species are associated with carcinogenesis of the pancreas, while some bacterial metabolites contribute to tumor-associated low-grade inflammation and immune responses via several proinflammatory factors and signaling pathways. Given the limited evidence on the interplay between gut microbiota and pancreatic cancer, risk factors associated with pancreatic cancer, such as diabetes, chronic pancreatitis and obesity, should also be taken into consideration. In terms of treatment of pancreatic cancer, gut microbiota has exhibited multiple effects on both traditional chemotherapy and the recently successful immunotherapy. Therefore, in this review, we summarize the latest developments and advancements in gut microbiota in relation to pancreatic cancer to elucidate its potential value.

Immune-Enhancing Activity of Aqueous Extracts from Artemisia rupestris L. via MAPK and NF-kB Pathways of TLR4/TLR2 Downstream in Dendritic Cells.

Artemisia rupestris L. has long been used as a traditional herbal medicine owing to its immunomodulatory activity. Aqueous extracts of Artemisia rupestris L. (AEAR) contain the main functional component and can activate the maturation of dendritic cells (DCs) and enhance the adaptive immunity as the adjuvant against infections. To explore the underlying mechanism of immunomodulatory activities of AEAR, DCs were produced from bone-marrow cells of mice and the effects of AEAR on cell viability were assessed by the Cell Counting Kit 8 (CCK8) method and annexin V/propidium iodide staining assays. Then, the effects of AEAR on the morphology, maturation, and function of DCs were detected using a microscope, flow cytometry-based surface receptor characterization, and endocytosis assays. The secretion levels of cytokines were then analyzed with enzyme-linked immunosorbent assay (ELISA). The activation state of DCs was evaluated by the mixed lymphocyte reaction (MLR). The activity of MAPKs and NF-κB pathways, which were involved in the regulation of AEAR on DCs, was further detected by Western blot. AEAR did not have a cytotoxic effect on DCs or mouse splenocytes. AEAR remarkably enhanced the phenotypic maturation of DCs and promoted the expression of costimulatory molecules and the secretion of cytokines in DCs. AEAR also significantly decreased the phagocytic ability of DCs and augmented the abilities of DCs to present antigens and stimulate allogeneic T-cell proliferation. Simultaneously, AEAR potently activated toll-like receptor (TLR)4-/TLR2-related MAPKs and induced the degradation of IκB and the translocation of NF-κB. In short, AEAR can profoundly enhance the immune-modulating activities of DCs via TLR4-/TLR2-mediated activation of MAPKs and NF-κB signaling pathways and is a promising candidate immunopotentiator for vaccines.

Hexosamine pathway inhibition overcomes pancreatic cancer resistance to gemcitabine through unfolded protein response and EGFR-Akt pathway modulation.

Different evidence has indicated metabolic rewiring as a necessity for pancreatic cancer (PC) growth, invasion, and chemotherapy resistance. A relevant role has been assigned to glucose metabolism. In particular, an enhanced flux through the Hexosamine Biosynthetic Pathway (HBP) has been tightly linked to PC development. Here, we show that enhancement of the HBP, through the upregulation of the enzyme Phosphoacetylglucosamine Mutase 3 (PGM3), is associated with the onset of gemcitabine (GEM) resistance in PC. Indeed, mRNA profiles of GEM sensitive and resistant patient-derived tumor xenografts (PDXs) indicate that PGM3 expression is specifically increased in GEM-resistant PDXs. Of note, PGM3 results also overexpressed in human PC tissues as compared to paired adjacent normal tissues and its higher expression in PC patients is associated with worse median overall survival (OS). Strikingly, genetic or pharmacological PGM3 inhibition reduces PC cell growth, migration, invasion, in vivo tumor growth and enhances GEM sensitivity. Thus, combined treatment between a specific inhibitor of PGM3, named FR054, and GEM results in a potent reduction of xenograft tumor growth without any obvious side effects in normal tissues. Mechanistically, PGM3 inhibition, reducing protein glycosylation, causes a sustained Unfolded Protein Response (UPR), a significant attenuation of the pro-tumorigenic Epidermal Growth Factor Receptor (EGFR)-Akt axis, and finally cell death. In conclusion this study identifies the HBP as a metabolic pathway involved in GEM resistance and provides a strong rationale for a PC therapy addressing the combined treatment with the PGM3 inhibitor and GEM.

Evaluation of aqueous extracts of Cistanche deserticola as a polysaccharide adjuvant for seasonal influenza vaccine in young adult mice.

Adjuvants can improve the efficacy of influenza vaccines and are still a hot spot in the study and development of influenza vaccines. In this report, the effects of aqueous extracts of Cistanche deserticola (AECD) as a polysaccharide adjuvant on seasonal influenza vaccines (IVV) were explored. The experimental data of anti-IVV IgG1 and IgG as well as hemagglutinin inhibition (HI) titers in young adult mice indicated that IVV intramuscularly co-injected with AECD was significantly more immunogenic than alum-adjuvanted or non-adjuvanted IVV. AECD-adjuvanted vaccine could rapidly initiate specific IgG response. Similarly, IVV with AECD augmented significantly lymphocyte proliferation and increased the positive rates of CD4+, CD8+ and CD44+ T cells from draining lymph nodes and spleens. Importantly, IVV with AECD could induce the Th1 immune response, as indicated by higher IgG2a levels accompanied by the induction of IFN-γ in CD4+ and CD8+ T cells. Additionally, IVV with AECD activated dendritic cells (DCs) and decreased the expression of Treg cells. There were no noticeable side effects after the vaccination. In brief, the addition of AECD enhanced immunogenicity to seasonal influenza vaccine by the induction of HI antibody generation, more rapid humoral immune responses, and a balanced Th1-/Th2-type response, effective T-cell responses, which may be important for seasonal influenza vaccines with broad and long lasting immunity.

Immunostimulatory activity of water-extractable polysaccharides from Cistanche deserticola as a plant adjuvant in vitro and in vivo.

A safe and effective vaccine adjuvant is important in modern vaccines. Various Chinese herbal polysaccharides can activate the immune system. Cistanche deserticola (CD) is a traditional Chinese herb and an adjuvant candidate. Here, we confirmed that water-extractable polysaccharides of CD (WPCD) could modulate immune responses in vitro and in vivo. In a dose-dependent manner, WPCD significantly promoted the maturation and function of murine marrow-derived dendritic cells (BM-DCs) through up-regulating the expression levels of MHC-II, CD86, CD80, and CD40, allogenic T cell proliferation, and the yields of IL-12 and TNF-α via toll-like receptor4 (TLR4), as indicated by in vitro experiments. In addition, its immunomodulatory activity was also observed in mice. WPCD effectively improved the titers of IgG, IgG1 and IgG2a and markedly enhanced the proliferation of T and B cells, the production of IFN-γ and IL-4 in CD4+ T cells and the expression level of IFN-γ in CD8+ T cells better than Alum. Furthermore, WPCD could markedly up-regulate the expression levels of CD40 and CD80 on DCs in spleen and down-regulate the Treg frequency. The study suggests that polysaccharides of Cistanche deserticola are a safe and effective vaccine adjuvant for eliciting both humoral immunity and cellular immunity by activating DCs via TLR4 signaling pathway.

NF-κB in pancreatic cancer: Its key role in chemoresistance.

Pancreatic cancer is considered a lethal disease with a high mortality and an extremely low five-year survival rate. Chemotherapy plays a pivotal role in pancreatic cancer treatment both in an adjuvant setting after complete resection and in the case of unresectable metastatic disease. However, none of the available combination chemotherapy regimens has resulted in satisfactory survival outcomes. Recent studies have revealed that both constitutive and induced activation of nuclear factor kappa B (NF-κB) in pancreatic cancer cells are closely associated with cell proliferation, invasion, anti-apoptosis, inflammation, angiogenesis and chemotherapeutic resistance. Therefore, NF-κB inhibitors in combination with cytotoxic compounds have been reported as novel agents that improve chemotherapy sensitivity in pancreatic cancer. In this review, we outline recent developments in the understanding of the role of the NF-κB signaling pathway and its associated genes in the progression of pancreatic cancer and highlight some potentially effective strategies for pancreatic cancer treatment.