Tumor Tissues Of Mice Research Articles

Comparison of the effects of probiotic strains (Lactobacillus gasseri, Lactiplantibacillus plantarum, Lactobacillus acidophilus, and Limosilactobacillus fermentum) isolated from human and food products on the immune response of CT26 tumor-bearing mice.

This study aimed to compare the effects of the probiotic bacteria, L. gasseri (52b), L. plantarum (M11), L. acidophilus (AC2), and L. fermentum (19SH), isolated from human source and traditional food products on the modulation of the immune system and inflammatory response on BALB/c mouse model bearing CT26 tumor. Five groups of female inbred BALB/c mice were orally administered with the probiotics and their mixes (MIX, at a 1:1 ratio) at varying dosages (1.5 × 108cfu/ml and 1.2 × 109cfu/ml) before and after the injection of a subcutaneous CT26 tumor over the course of 38days via gavage. Finally, their effects on the tumor apoptosis and the cytokine levels in spleen cell cultures were analyzed and compared. M11, MIX, and 52b groups had the greatest levels of interleukin-12 (IL-12) and interferon gamma (IFN-γ) production. The highest production level of granzyme B (GrB) was related to the MIX and 52b groups. Moreover, these groups showed the lowest production level of (IL-4) and transforming growth factor beta (TGF-β). Furthermore, the groups of MIX and 52b demonstrated the greatest amount of lymphocyte proliferation of spleen cells in response to the tumor antigen. The delayed-type hypersensitivity (DTH) response significantly increased in the groups of MIX and 52b compared with the control (p < 0.05). The findings demonstrated that the oral treatment of the human strain (52b) and the combination of these bacteria generated strong T helper type 1 (Th1) immune responses in the tumor tissue of the tumor-bearing mice, which led to the suppression of the tumor development.

Environmental cadmium exposure facilitates mammary tumorigenesis via reprogramming gut microbiota-mediated glutamine metabolism in MMTV-Erbb2 mice

Cadmium (Cd) is a heavy metal that has been widely reported to be linked to the onset and progression of breast cancer (BC). However, the mechanism of Cd-induced mammary tumorigenesis remains elusive. In our study, a transgenic mouse model that spontaneously develops tumors through overexpression of wild-type Erbb2 (MMTV-Erbb2) was constructed to investigate the effects of Cd exposure on BC tumorigenesis. The results showed that oral exposure to 3.6 mg/L Cd for 23 weeks dramatically accelerated tumor appearance and growth, increased Ki67 density and enhanced focal necrosis and neovascularization in the tumor tissue of MMTV-Erbb2 mice. Notably, Cd exposure enhanced glutamine (Gln) metabolism in tumor tissue, and 6-diazo-5-oxo-l-norleucine (DON), a Gln metabolism antagonist, inhibited Cd-induced breast carcinogenesis. Then our metagenomic sequencing and mass spectrometry-based metabolomics confirmed that Cd exposure disturbed gut microbiota homeostasis, especially Helicobacter and Campylobacter abundance remodeling, which altered the gut metabolic homeostasis of Gln. Moreover, intratumoral Gln metabolism profoundly increased under Cd-elevated gut permeability. Importantly, depletion of microbiota with an antibiotic cocktail (AbX) treatment led to a significant delay in the appearance of palpable tumors, inhibition of tumor growth, decrease in tumor weight, reduction in Ki67 expression and low-grade pathology in Cd-exposed MMTV-Erbb2 mice. Also, transplantation of Cd-modulated microbiota decreased tumor latency, accelerated tumor growth, increased tumor weight, upregulated Ki67 expression and exacerbated neovascularization as well as focal necrosis in MMTV-Erbb2 mice. In summary, Cd exposure induced gut microbiota dysbiosis, elevated gut permeability and increased intratumoral Gln metabolism, leading to the promotion of mammary tumorigenesis. This study provides novel insights into environmental Cd exposure-mediated carcinogenesis.

Jinfukang inhibits lung cancer metastasis by regulating T cell receptors

Ethnopharmacological relevanceMetastasis is the leading cause of death in lung cancer worldwide, and immune escape plays a vital role in the process of metastasis. Clinical studies have proven that Jinfukang (JFK) can effectively treat lung cancer metastasis by regulating T lymphocytes. However, it is still unknown whether JFK plays a role in treating lung cancer metastasis by regulating T-cell receptors (TCRs). Aim of the studyTo explore the effect of JFK in inhibiting lung cancer metastasis by regulating TCR. Materials and methodsA lung metastasis model was established in C57BL/6J and BALB/c-nude mice by tail vein injection of Lewis lung cancer cells. JFK was given by continuous intragastric administration. Anatomical observation combined with hematoxylin-eosin staining was used to evaluate lung metastasis. T cells, MDSCs, and macrophages in the peripheral blood were detected by flow cytometry, and the proliferation and immune cell infiltration of lung metastases were observed by immunohistochemistry and immunofluorescence. The diversity and gene expression of TCR in peripheral blood and lung tissues were detected by immune repertoire sequencing, and bioinformatics analysis was carried out. ResultsCompared with the control group, the number of pulmonary metastatic nodules in JFK-treated mice showed a decreasing trend, and it significantly reduced the burden of lung tumor metastasis in mice. We found that the expression level of Ki-67 protein in lung metastatic tumor tissues of mice treated with JFK was significantly reduced, while the infiltration level of CD8+ T lymphocytes and NK cells was significantly increased. In addition, we also found that JFK could significantly increase the proportion of CD4+ T, CD8+ T and NKT cells in the peripheral blood of mice. Moreover, JFK reduced the ratio of M-MDSCs and increased the ratio of PMN-MDSCs in the peripheral blood of mice. JFK increased the ratio of M1 macrophages in the peripheral blood of Lewis tumor-bearing mice. The sequencing of TCR in the peripheral blood and lung tissue of mice indicated that there was no notable difference in TCR diversity as the tumor progressed and JFK treatment was administered. However, the downregulation of TRBV16, TRBV17, TRBV1 and the upregulation of the TRBV12-2 gene in the TCR caused by tumor progression can be reversed by JFK. ConclusionThese results suggest that JFK may upregulate the proportion of CD4+ T, CD8+ T and NKT cells in peripheral blood, reverse the TCR changes caused by tumor metastasis, and promote the infiltration of CD8+ T and NK cells in tumor tissues, thereby inhibiting the growth of tumors and ultimately reducing the burden of lung cancer metastasis. This will provide new strategies for developing Chinese herbal medicine to treat metastasis by regulating TCR.

Overexpression of SERPINA3 suppresses tumor progression by modulating SPOP/NF‑κB in lung cancer.

The pathogenesis mechanism of lung cancer is very complex, with high incidence and mortality. Serpin family A member 3 (SERPINA3) expression levels were reduced in the sera of patients with lung cancer and may be a candidate diagnostic and prognostic survival biomarker in lung cancer, as previously reported. However, the detailed biological functions of SERPINA3 in the pathogenesis of lung cancer remain unknown. In the present study, it was aimed to explore the effects of SERPINA3 on the occurrence of lung cancer. SERPINA3 expression was assessed using bioinformatics database analysis and experimental detection. Then, the biological effects of SERPINA3 were investigated in a cell culture system and a xenograft model of human lung cancer. The potential regulatory mechanism of SERPINA3 in lung cancer was explored by data‑independent acquisition mass spectrometry (DIA‑MS) detection and further validated by western blotting (WB). The results indicated that SERPINA3 expression levels were significantly downregulated in lung cancer tissues and cell lines. At the cellular level, it was revealed that overexpressed SERPINA3 inhibited cell growth, proliferation, migration and invasion and promoted the apoptosis of lung cancer cells. Moreover, overexpressed SERPINA3 enhanced the sensitivity of lung cancer cells to osimertinib. Invivo, a xenograft model of human lung cancer was established with BALB/c nude mice. After the injection of A549 cells, the tumor growth of the tumor‑bearing mice in the SERPINA3‑overexpressing group increased more slowly, and the tumor volume was smaller than that in the empty‑vector group. Mechanistically, a total of 65 differentially expressed proteins were identified. It was found that the speckle‑type POZ protein (SPOP) was significantly upregulated in SERPINA3‑overexpressing H157 cells using DIA‑MS detection and analysis. WB validation showed that SPOP expression increased, and NF‑kappaB (NF‑κB) p65 was inhibited in cell lines and tumor tissues of mice when SERPINA3 was overexpressed. The present findings suggest that SERPINA3 is involved in the development of lung cancer and has an antineoplastic role in lung cancer.

Discovery of ARD-2051 as a Potent and Orally Efficacious Proteolysis Targeting Chimera (PROTAC) Degrader of Androgen Receptor for the Treatment of Advanced Prostate Cancer.

We report the discovery of ARD-2051 as a potent and orally efficacious androgen receptor (AR) proteolysis-targeting chimera degrader. ARD-2051 achieves DC50 values of 0.6 nM and Dmax >90% in inducing AR protein degradation in both the LNCaP and VCaP prostate cancer cell lines, potently and effectively suppresses AR-regulated genes, and inhibits cancer cell growth. ARD-2051 achieves a good oral bioavailability and pharmacokinetic profile in mouse, rat, and dog. A single oral dose of ARD-2051 strongly reduces AR protein and suppresses AR-regulated gene expression in the VCaP xenograft tumor tissue in mice. Oral administration of ARD-2051 effectively inhibits VCaP tumor growth and causes no signs of toxicity in mice. ARD-2051 is a promising AR degrader for advanced preclinical development for the treatment of AR+ human cancers.

CHI3L1 induces autophagy through the JNK pathway in lung cancer cells

CHI3L1 is closely related to the molecular mechanisms of cancer cell migration, growth, and death. According to recent research, autophagy regulates tumor growth during various stages of cancer development. This study examined the association between CHI3L1 and autophagy in human lung cancer cells. In CHI3L1-overexpressing lung cancer cells, the expression of LC3, an autophagosome marker, and the accumulation of LC3 puncta increased. In contrast, CHI3L1 depletion in lung cancer cells decreased the formation of autophagosomes. Additionally, CHI3L1 overexpression promoted the formation of autophagosomes in various cancer cell lines: it also increased the co-localization of LC3 and the lysosome marker protein LAMP-1, indicating an increase in the production of autolysosomes. In mechanism study, CHI3L1 promotes autophagy via activation of JNK signaling. JNK may be crucial for CHI3L1-induced autophagy since pretreatment with the JNK inhibitor reduced the autophagic effect. Consistent with the in vitro model, the expression of autophagy-related proteins was downregulated in the tumor tissues of CHI3L1-knockout mice. Furthermore, the expression of autophagy-related proteins and CHI3L1 increased in lung cancer tissues compared with normal lung tissues. These findings show that CHI3L1-induced autophagy is triggered by JNK signals and that CHI3L1-induced autophagy could be a novel therapeutic approach to lung cancer.

Delivery of SIRT1 by cancer-associated adipocyte-derived extracellular vesicles regulates immune response and tumorigenesis of ovarian cancer cells.

This study intends to investigate the possible molecular mechanism of immune response and tumorigenesis in ovarian cancer cells, mediated by sirtuin 1 (SIRT1)-containing extracellular vesicles (EVs) derived from cancer-associated adipocytes (CAAs) (CAA-EVs). Differentially expressed genes in EVs from CAAs were screened by RNA transcriptome sequencing, and the downstream pathway was predicted in silico. The binding between SIRT1 and CD24 was investigated by luciferase activity and ChIP-PCR assays. EVs were extracted from human ovarian cancer tissue-isolated CAAs, and the internalization of CCA-EVs by ovarian cancer cells was characterized. The ovarian cancer cell line was injected into mice to establish an animal model. Flow cytometry was performed to analyze the proportions of M1 and M2 macrophages, CD8+ T, T-reg, and CD4+ T cells. TUNEL staining was used to detect cell apoptosis in the mouse tumor tissues. ELISA detection was performed on immune-related factors in the serum of mice. CAA-EVs could deliver SIRT1 to ovarian cancer cells, thereby affecting the immune response of ovarian cancer cells in vitro and promoting tumorigenesis in vivo. SIRT1 could transcriptionally activate the expression of CD24, and CD24 could up-regulate Siglec-10 expression. CAA-EVs-SIRT1 activated the CD24/Siglec-10 axis and promoted CD8+ T cell apoptosis, thereby promoting tumorigenesis in mice. CAA-EVs-mediated transfer of SIRT1 regulates the CD24/Siglec-10 axis to curb immune response and promote tumorigenesis of ovarian cancer cells.

Phenotyping of lymphoproliferative tumours generated in xenografts of non-small cell lung cancer.

Patient-derived xenograft (PDX) models involve the engraftment of tumour tissue in immunocompromised mice and represent an important pre-clinical oncology research method. A limitation of non-small cell lung cancer (NSCLC) PDX model derivation in NOD-scid IL2Rgammanull (NSG) mice is that a subset of initial engraftments are of lymphocytic, rather than tumour origin. The immunophenotype of lymphoproliferations arising in the lung TRACERx PDX pipeline were characterised. To present the histology data herein, we developed a Python-based tool for generating patient-level pathology overview figures from whole-slide image files; PATHOverview is available on GitHub (https://github.com/EpiCENTR-Lab/PATHOverview). Lymphoproliferations occurred in 17.8% of lung adenocarcinoma and 10% of lung squamous cell carcinoma transplantations, despite none of these patients having a prior or subsequent clinical history of lymphoproliferative disease. Lymphoproliferations were predominantly human CD20+ B cells and had the immunophenotype expected for post-transplantation diffuse large B cell lymphoma with plasma cell features. All lymphoproliferations expressed Epstein-Barr-encoded RNAs (EBER). Analysis of immunoglobulin light chain gene rearrangements in three tumours where multiple tumour regions had resulted in lymphoproliferations suggested that each had independent clonal origins. Overall, these data suggest that B cell clones with lymphoproliferative potential are present within primary NSCLC tumours, and that these are under continuous immune surveillance. Since these cells can be expanded following transplantation into NSG mice, our data highlight the value of quality control measures to identify lymphoproliferations within xenograft pipelines and support the incorporation of strategies to minimise lymphoproliferations during the early stages of xenograft establishment pipelines.

Systemic administration of mesenchymal stem cells loaded with a novel oncolytic adenovirus carrying a bispecific T cell engager against hepatocellular carcinoma

ABSTRACT We previously established a hepatocellular carcinoma (HCC) targeting system of conditionally replicative adenovirus (CRAd) delivered by human umbilical cord-derived mesenchymal stem cells (HUMSCs). However, this system needed to be developed further to enhance the antitumor effect and overcome the limitations caused by the alpha-fetoprotein (AFP) heterogeneity of HCC. In this study, a bispecific T cell engager (BiTE) targeting programmed death ligand 1 controlled by the human telomerase reverse transcriptase promoter was armed on the CRAd of the old system. It was demonstrated on orthotopic transplantation model mice that the new system had a better anti-tumor effect with no more damage to extrahepatic organs and less liver injury, and the infiltration and activation of T cells were significantly enhanced in the tumor tissues of the model mice treated with the new system. Importantly, we confirmed that the new system eliminated the AFP-negative cells on AFP heterogeneous tumor models efficiently. Conclusion: Compared with the old system, the new system provided a more effective and safer strategy against HCC.

Effect of doxorubicin on efficacy of immune checkpoint inhibitors through increasing infiltration of CD8-positive T cells and activating STAT1-IRF1-CXCL10 axis.

e13095 Background: Immune checkpoint inhibitors (ICIs) generate an enduring clinical remission in metastatic cancers. However, the efficacy of ICIs monotherapy for triple negative breast cancer (TNBC) is limited. Strategies are urgently needed to enhance anticancer immune responses in TNBC to prolong survival of patients. Methods: BALB/c mice subcutaneous tumor experiments were done to evaluate the efficacy of doxorubicin combined with PD-1 inhibitor, compared with other chemotherapy including cisplatin and paclitaxel. RNA-seq and single-sample gene set enrichment analysis (ssGSEA) were done with our collected mice tumor tissue to observe changes of tumor immune environment in TNBC after chemotherapy combined with immunotherapy. Then, we performed coculture, Transwell assay and FACs in TNBC cell lines, including MDA-MB-231, BT549 and 4T1, and primary CD8+T cells, to assess effects of doxorubicin on CD8+T cell migration in vitro. Gene Ontology (GO) and Kyoto Encyclopedia of Genes, Genomes (KEGG) analysis were enriched from RNA-seq data to seek for a pathway. We analyzed the difference of CD8+T cell infiltration related gene expression, including CCL2, CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11. Western blot, qPCR and ELISA were utilized to explore possible mechanism in stronger migration of CD8+T cells by doxorubicin. Rescue assays were used to confirm the involved mechanisms. Results: Subcutaneous tumor experiment showed that doxorubicin could enhance the efficacy of immune checkpoint inhibitors (p＜0.05),andthe immune microenvironment in TNBC transferred from "anti-inflammatory" to "proinflammatory" status after doxorubicin combined with immunotherapy, compared with other chemotherapy drugs combined with immunotherapy. Higher CD8+T cells infiltration occurred in TNBC treated with doxorubicin in vivo (p＜0.05). The CD8+T cells migration assay demonstrated a higher CD8+T cell migration with doxorubicin in vitro (p＜0.05). KEGG analysis revealed that the chemokine-related pathway was significantly activated after doxorubicin treatment, in which CXCL10 chemokine was most likely to be responsible for CD8+T cell infiltration based on its expression and secretion (both p&lt;0.01). Further experiments demonstrated that the expression of the whole STAT1-IRF1-CXCL10 axis was up-regulated in TNBC mice treated with doxorubicin (p＜0.01), and the STAT1-IRF1-CXCL10 axis was also activated in doxorubicin-treated TNBC cell lines (p＜0.0001). Conclusions: This research demonstrated that doxorubicin can enhance efficacy of immune checkpoint inhibitors through increasing infiltration of CD8+T cells in triple-negative breast cancer via activating STAT1-IRF1-CXCL10 axis. These results provide rationales of doxorubicin combined with PD-1 inhibitor for extending survival of TNBC patients.

Mechanistic insights into the anti-tumor and anti-metastatic effects of Patrinia villosa aqueous extract in colon cancer via modulation of TGF-β R1-smad2/3-E-cadherin and FAK-RhoA-cofilin pathways

BackgroundPatrinia villosa, a traditional medicinal herb commonly used for treating intestinal-related diseases, has been commonly prescribed by Chinese medicine practitioners as a key component herb to treat colon cancer, although its anti-tumor effect and mechanisms of action have not been fully elucidated. Hypothesis/purposeThis study aimed to investigate the anti-tumor and anti-metastatic effects of Patrinia villosa aqueous extract (PVW), and its underlying mechanisms. MethodThe chemical profile of PVW was analysed by high-performance liquid chromatography with photodiode-array detection (HPLC-DAD) method. Cell-based functional assays MTT, BrdU, scratch, and transwell were conducted to evaluate the effects of PVW on human colon cancer HCT116 and murine colon26-luc cells, assessing cytotoxicity, cell proliferation, motility, and migration, respectively. Western blotting was performed to assess the effect of PVW on the expression of key intracellular signaling proteins. In vivo studies were conducted using zebrafish embryos and tumor-bearing mice to evaluate the anti-tumor, anti-angiogenesis, and anti-metastatic effects of PVW in colon cancer. ResultsFive chemical markers were identified and quantified in PVW. PVW exhibited significant cytotoxicity and anti-proliferative activity, as well as inhibitory effects on cell motility and migration in both HCT116 and colon 26-luc cancer cells via modulating protein expressions of TGF-β R1, smad2/3, snail, E-cadherin, FAK, RhoA, and cofilin. PVW (0.01–0.1 mg/ml) could significantly decrease the length of subintestinal vessels of zebrafish embryos through decreasing mRNA expressions of FLT1, FLT4, KDRL, VEGFaa, VEGFc, and Tie1. PVW (> 0.05 mg/ml) also significantly suppressed colon cancer cells migration in the zebrafish embryos. Furthermore, oral administration of PVW (1.6 g/kg) significantly inhibited tumor growth by decreasing the expressions of tumor activation marker Ki-67 and CD 31 in tumor tissues of HCT116 tumor-bearing mice. PVW could also significantly inhibit lung metastasis in colon 26-luc tumor-bearing mice by modulating their tumor microenvironment, including immune cells populations (T cells and MDSCs), levels of cytokines (IL-2, IL-12, and IFN-γ), as well as increasing the relative abundance of gut microbiota. ConclusionThis study revealed for the first time the anti-tumor and anti-metastatic effects of PVW through regulation of TGF-β-smad2/3-E-cadherin, and FAK-cofilin pathways in colon cancer. These findings provide scientific evidence to support the clinical use of P. villosa in patients with colon cancer.

Establishment of a novel co-cultured liver cancer model based on tumor microenvironment

Objective: To construct a new co-cultured liver cancer research model composed of activated hepatic stellate cells (aHSC) and liver cancer cells, explore the efficacy difference between it and traditional model, so as to establish a liver cancer research model in vitro and in vivo that can reflect the real clinical efficacy. Methods: A new co-culture model of liver cancer consisting of aHSC and liver cancer cells was constructed. The differences in efficacy between the new co-culture model and the traditional single cell model were compared by cytotoxicity test, cell migration test, drug retention test and in vivo tumor inhibition test. Western blot was used to detect the drug-resistant protein P-gp and epithelial-mesenchymal transition-related proteins. Masson staining was used to observe the deposition of collagen fibers in tumor tissues of tumor-bearing mice. CD31 immunohistochemical staining was used to observe the microvessel density in tumor tissues of tumor-bearing mice. Results: The cytotoxicity of single cell model and co-culture model was dose-dependent. With the increase of curcumin (CUR) concentration, the cell viability decreased, but the cell viability of single cell model decreased faster than that of co-culture model. When the concentration of CUR was 10 μg/ml, the cell viability of the co-culture model was 62.3% and the migration rate was (28.05±3.68)%, which were higher than those of the single cell model [38.5% and (14.91±5.92)%, both P<0.05]. Western blot analysis showed that the expressions of P-gp and vimentin were up-regulated in the co-culture model, which were 1.55 and 2.04 fold changes of the single cell model, respectively. The expression of E-cadherin was down-regulated, and the expression level of E-cadherin in the single cell model was 1.17 fold changes of the co-culture model. Drug retention experiment showed that the co-culture model could promote drug efflux and reduce drug retention. In vivo tumor inhibition experiment showed that the m-HSC+ H22 co-transplantation model had faster tumor growth and larger tumor volume than those of the H22 single cell transplantation model. After CUR treatment, the tumor growths of m-HSC+ H22 co-transplantation model and H22 single cell transplantation model were inhibited. Masson staining showed that the deposition of collagen fibers in tumor tissues of m-HSC+ H22 co-transplantation model mice was more than that of H22 single cell transplantation model. CD31 immunohistochemical staining showed that the microvessel density in tumor tissue of m-HSC+ H22 co-transplantation model was higher than that of H22 single cell transplantation model. Conclusions: The aHSC+ liver cancer cell co-culture model has strong proliferation and metastasis ability and is easy to be resistant to drugs. It is a new type of liver cancer treatment research model superior to the traditional single cell model.

Synthesis and Evaluation of Clinically Translatable Targeted Microbubbles Using a Microfluidic Device for In Vivo Ultrasound Molecular Imaging.

The main aim of this study is to synthesize contrast microbubbles (MB) functionalized with engineered protein ligands using a microfluidic device to target breast cancer specific vascular B7-H3 receptor in vivo for diagnostic ultrasound imaging. We used a high-affinity affibody (ABY) selected against human/mouse B7-H3 receptor for engineering targeted MBs (TMBs). We introduced a C-terminal cysteine residue to this ABY ligand for facilitating site-specific conjugation to DSPE-PEG-2K-maleimide (M. Wt = 2.9416 kDa) phospholipid for MB formulation. We optimized the reaction conditions of bioconjugations and applied it for microfluidic based synthesis of TMBs using DSPE-PEG-ABY and DPPC liposomes (5:95 mole %). The binding affinity of TMBs to B7-H3 (MBB7-H3) was tested in vitro in MS1 endothelial cells expressing human B7-H3 (MS1B7-H3) by flow chamber assay, and by ex vivo in the mammary tumors of a transgenic mouse model (FVB/N-Tg (MMTV-PyMT)634Mul/J), expressing murine B7-H3 in the vascular endothelial cells by immunostaining analyses. We successfully optimized the conditions needed for generating TMBs using a microfluidic system. The synthesized MBs showed higher affinity to MS1 cells engineered to express higher level of hB7-H3, and in the endothelial cells of mouse tumor tissue upon injecting TMBs in a live animal. The average number (mean ± SD) of MBB7-H3 binding to MS1B7-H3 cells was estimated to be 354.4 ± 52.3 per field of view (FOV) compared to wild-type control cells (MS1WT; 36.2 ± 7.5/FOV). The non-targeted MBs did not show any selective binding affinity to both the cells (37.7 ± 7.8/FOV for MS1B7-H3 and 28.3 ± 6.7/FOV for MS1WT cells). The fluorescently labeled MBB7-H3 upon systemic injection in vivo co-localized to tumor vessels, expressing B7-H3 receptor, as validated by ex vivo immunofluorescence analyses. We have successfully synthesized a novel MBB7-H3 via microfluidic device, which allows us to produce on demand TMBs for clinical applications. This clinically translatable MBB7-H3 showed significant binding affinity to vascular endothelial cells expressing B7-H3 both in vitro and in vivo, which shows its potential for clinical translation as a molecular ultrasound contrast agent for human applications.

Marsdenia tenacissima extract induces endoplasmic reticulum stress-associated immunogenic cell death in non-small cell lung cancer cells through targeting AXL

Ethnopharmacological relevanceMarsdenia Tenacissima (Roxb.) Wight et Arn. is a traditional Chinese medicine. Its standardized extract (MTE), with the trade name Xiao-Ai-Ping injection, is widely used for cancer treatment. The pharmacological effects of MTE-inducing cancer cell death have been primarily explored. However, whether MTE triggers tumor endoplasmic reticulum stress (ERS)-associated immunogenic cell death (ICD) is unknown. Aim of the studyTo determine the potential role of endoplasmic reticulum stress in the anti-cancer effects of MTE, and uncover the possible mechanisms of endoplasmic reticulum stress-associated immunogenic cell death induced by MTE. Material and methodsThe anti-tumor effects of MTE on non-small cell lung cancer (NSCLC) were examined through CCK-8 and wound healing assay. Network pharmacology analysis and RNA-sequencing (RNA seq) were performed to confirm the biological changes of NSCLCs after MTE treatment. Western blot, qRT-PCR, reactive oxygen species (ROS) assay, and mitochondrial membrane potential (MMP) assay were used to explore the occurrence of endoplasmic reticulum stress. Immunogenic cell death-related markers were tested by ELISA and ATP release assay. Salubrinal was used to inhibit the endoplasmic reticulum stress response. SiRNA and bemcentinib (R428) were used to impede the function of AXL. AXL phosphorylation was regained by recombinant human Gas6 protein (rhGas6). The effects of MTE on endoplasmic reticulum stress and immunogenic cell death response were also proved in vivo. The AXL inhibiting compound in MTE was explored by molecular docking and confirmed by Western blot. ResultsMTE inhibited cell viability and migration of PC-9 and H1975 cells. Enrichment analysis identified that differential genes after MTE treatment were significantly enriched in endoplasmic reticulum stress-related biological processes. MTE decreased mitochondrial membrane potential (MMP) and increased ROS production. Meanwhile, endoplasmic reticulum stress-related proteins (ATF6, GRP-78, ATF4, XBP1s, and CHOP) and immunogenic cell death-related markers (ATP, HMGB1) were upregulated, and the AXL phosphorylation level was suppressed after MTE treatment. However, when salubrinal (an endoplasmic reticulum stress inhibitor) and MTE were co-treated cells, the inhibitory effects of MTE on PC-9 and H1975 cells were impaired. Importantly, inhibition of AXL expression or activity also promotes the expression of endoplasmic reticulum stress and immunogenic cell death-related markers. Mechanistically, MTE induced endoplasmic reticulum stress and immunogenic cell death by suppressing AXL activity, and these effects were attenuated when AXL activity recovered. Moreover, MTE significantly increased the expression of endoplasmic reticulum stress-related markers in LLC tumor-bearing mouse tumor tissues and plasma levels of ATP and HMGB1. Molecular docking illustrated that kaempferol has the strongest binding energy with AXL and suppresses AXL phosphorylation. ConclusionMTE induces endoplasmic reticulum stress-associated immunogenic cell death in NSCLC cells. The anti-tumor effects of MTE are dependent upon endoplasmic reticulum stress. MTE triggers endoplasmic reticulum stress-associated immunogenic cell death by inhibiting AXL activity. Kaempferol is an active component that inhibits AXL activity in MTE. The present research revealed the role of AXL in regulating endoplasmic reticulum stress and enriched the anti-tumor mechanisms of MTE. Moreover, kaempferol may be considered a novel AXL inhibitor.

Circ_0003356 suppresses gastric cancer growth through targeting the miR-668-3p/SOCS3 axis.

Circular RNAs (circRNAs) have attracted extensive attention as therapeutic targets in gastric cancer (GC). Circ_0003356 is known to be downregulated in GC tissues, but its cellular function and mechanisms remain undefined. To investigate the role of circ_0003356 in GC at the molecular and cellular level. Circ_0003356, miR-668-3p, and SOCS3 expression were assessed via quantitative real time-polymerase chain reaction (qRT-PCR). Wound healing, EdU, CCK-8, flow cytometry and transwell assays were used to analyze the migration, proliferation, viability, apoptosis and invasion of GC cells. The subcellular localization of circ_0003356 was monitored using fluorescence in situ hybridization. The interaction of circ_0003356 with miR-668-3p was confirmed using RIP-qRT-PCR, RNA pull-down, and dual luciferase reporter assays. We observed protein levels of genes via western blot. We injected AGS cells into the upper back of mice and performed immunohistochemistry staining for examining E-cadherin, N-cadherin, Ki67, and SOCS3 expressions. TUNEL staining was performed for the assessment of apoptosis in mouse tumor tissues. Circ_0003356 and SOCS3 expression was downregulated in GC cells, whilst miR-668-3p was upregulated. Exogenous circ_0003356 expression and miR-668-3p silencing suppressed the migration, viability, proliferation, epithelial to mesenchy-mal transition (EMT) and invasion of GC cells and enhanced apoptosis. Circ_0003356 overexpression impaired tumor growth in xenograft mice. Targeting of miR-668-3p by circ_0003356 was confirmed through binding assays and SOCS3 was identified as a downstream target of miR-668-3p. The impacts of circ_0003356 on cell proliferation, apoptosis, migration, invasion and EMT were reversed by miR-668-3p up-regulation or SOCS3 down-regulation in GC cells. Circ_0003356 impaired GC development through its interaction with the miR-668-3p/SOCS3 axis.

Identifying spatial immuno-oncological parameters of mouse tumors using Imaging Mass Cytometry

Abstract Identification of immuno-oncological (IO) processes that dictate tumor growth, metastasis, and immune response is essential for selecting promising drug candidates in preclinical models. Imaging Mass Cytometry™ (IMC™) is a proven high-plex imaging technology that enables deep characterization of the complexity of tumor tissue without disrupting spatial context while eliminating artifacts caused by tissue autofluorescence. Here, we showcase the Maxpar® OnDemand™ Mouse Immuno-Oncology IMC Panel Kit for application on mouse tumor tissues. We compiled a 33-plex antibody panel to quantitatively assess IO-related processes and applied it to a tissue microarray containing a large variety of mouse tumors. We digitized high-plex data from mouse tissues using the Hyperion™ Imaging System and generated images demonstrating the detailed layout of the tumor microenvironment (TME). We further conducted single-cell analysis to identify specific populations of tumor and immune cells in the TME. The Mouse IO IMC Panel Kit successfully identified pathophysiological processes such as immune cell infiltration and activation, signaling pathway activation, biomarkers of epithelial-to-mesenchymal transition (EMT), metabolic activity, growth, and the TME tissue architecture. Single-cell analysis of several highly relevant tumor types separated distinct cellular clusters representing tumor, immune, stromal, and vascular cells. Our quantitative analysis of tumor composition revealed critical insights regarding prognostic parameters such as metastatic and growth potential of tumor cells and activity of immune cell infiltrates. For Research Use Only. Not for use in diagnostic procedures.

Semiquantitative Analysis for High-Speed Mapping Applications of Biological Samples Using LA-ICP-TOFMS.

Laser ablation (LA) in combination with inductively coupled plasma time-of-flight mass spectrometry (ICP-TOFMS) enables monitoring of elements from the entire mass range for every pixel, regardless of the isotopes of interest for a certain application. This provides nontargeted multi-element (bio-)imaging capabilities and the unique possibility to screen for elements that were initially not expected in the sample. Quantification of a large range of elements is limited as the preparation of highly multiplexed calibration standards for bioimaging applications by LA-ICP-(TOF)MS is challenging. In this study, we have developed a workflow for semiquantitative analysis by LA-ICP-TOFMS based on multi-element gelatin micro-droplet standards. The presented approach is intended for the mapping of biological samples due to the requirement of matrix-matched standards for accurate quantification in LA-ICPMS, a prerequisite that is given by the use of gelatin-based standards. A library of response factors was constructed based on 72 elements for the semiquantitative calculations. The presented method was evaluated in two stages: (i) on gelatin samples with known elemental concentrations and (ii) on real-world samples that included prime examples of bioimaging (mouse spleen and tumor tissue). The developed semiquantification approach was based on 10 elements as calibration standards and provided the determination of 136 nuclides of 63 elements, with errors below 25%, and for half of the nuclides, below 10%. A web application for quantification and semiquantification of LA-ICP(-TOF)MS data was developed, and a detailed description is presented to easily allow others to use the presented method.

Surface engineering of hollow gold nanoparticle with mesenchymal stem cell membrane and MUC-1 aptamer for targeted theranostic application against metastatic breast cancer

Mesenchymal stem cell membrane (MSCM)-coated biomimetic doxorubicin-loaded hollow gold nanoparticles were fabricated and decorated with MUC1 aptamer in order to provide smart theranostic platform. The prepared targeted nanoscale biomimetic platform was extensively characterized and evaluated in terms of selective delivery of DOX and CT-scan imaging.The fabricated system illustrated spherical morphology with 118 nm in diameter. Doxorubicin was loaded into the hollow gold nanoparticles through physical absorption technique with encapsulation efficiency and loading content of 77%±10 and 31%±4, respectively. The in vitro release profile demonstrated that the designed platform could respond to acidic environment, pH 5.5 and release 50% of the encapsulated doxorubicin during 48 h, while 14% of the encapsulated doxorubicin was released in physiological condition, pH 7.4 up to 48 h. The in vitro cytotoxicity experiments on 4T1 as MUC1 positive cell line illustrated that the targeted formulation could significantly increase mortality at 0.468 and 0.23 µg/ml of equivalent DOX concentration compared to non-targeted formulation while this cytotoxicity was not observed in CHO as MUC1 negative cell line. Furthermore, in vivo experiments showed high tumor accumulation of the targeted formulation even 24 h after intravenous injection which induced effective tumor growth suppression against 4T1 tumor bearing mice. On the other hand, existence of hollow gold in this platform provided CT scan imaging capability of the tumor tissue in 4T1 tumor bearing mice up to 24 h post-administration. The obtained results indicated that the designed paradigm are promising and safe theranostic system for fighting against metastatic breast cancer.

Effect of Ethanol-Induced Methyl Donors Consumption on the State of Hypomethylation in Cervical Cancer.

Cervical cancer causes malignant tumors in females and threatens the physical and mental health of women. Current research shows that persistent infection of high-risk HPV is the main cause of cervical cancer. However, not all cervical cancer is caused by HPV infection, which may also be related to other factors, such as nutritional status and lifestyle. This study focuses on the effect of alcohol consumption on the methylation status of cervical cancer from the perspective of methyl donors. We established a mouse tumor-bearing model with cervical cancer SiHa cells, and at the same time, we cultured SiHa cells in vitro. Different concentrations of ethanol were administered to the model mice and SiHa cells. Then, we detected the levels of the methyl-donor folate and methionine and their metabolite homocysteine levels in mice serum, tumor tissues, and SiHa cells. Furthermore, we determined the expression of the members of the DNA methyltransferase family (DNMT1, DNMT3a, and DNMT3b) in tumor tissue by immunohistochemistry. qRT-PCR and Western blotting analysis were used to detect the mRNA and protein levels of members of the DNA methyltransferase family in cervical cancer SiHa cells. Our results show that the levels of the methyl donor (folate and methionine) decreased with the increase of ethanol concentration (p < 0.05), and the homocysteine level increased significantly (p < 0.05). In SiHa cells, the mRNA and protein levels of the DNMT family members and their receptors were significantly higher than those in the control group (p < 0.05). Collectively, these results suggest that ethanol could influence DNMT expression by inducing methyl donor consumption, thereby causing cervical cancer cells to exhibit genome-wide hypomethylation.

Multi salt strategy based on curcumin pyrimidine derivatives prodrugs: Synthesis, biological activity, in vitro and in vivo imaging, and drug distribution research

Herein, a feasible strategy to enhance the water solubility of potential anti-tumor drugs was utilized, which was to combine hydrochloric acid or sulfuric acid into its molecules. In order to increase drug targeting, three different types of Eudragit® (stomach soluble, enteric soluble and permeable) was used to encapsulate these two salt under mild reaction conditions to obtain six kinds of nano polymer complexes. In order to simulate the gastrointestinal environment, spectroscopic tests were conducted on nanoparticles in PBS buffer solutions with different pH values. The micromorphology of the six complexes was analyzed by TEM. The results showed that the potential drugs were uniformly dispersed in the drug carrier materials, and the particle size was 100–500 nm. The Zeta potential test showed that the complexes had positive stability. The confocal microscope imaging experiments were carried out in different channels of HCT-116 and LO2 cells. The results showed that the complexes had favorable biocompatibility and imaging properties. The calculation of Pearson coefficient showed that some complexes could enter the nucleus. The cell proliferation toxicity test (CCK8) experiment and the TEM experiment of cell uptake localization showed that the complex had definite anti-tumor activity and low cytotoxicity, and the distribution of the complexes was detected in the nucleus and cytoplasm. In vivo imaging experiment, strong fluorescence intensity of the compounds was detected in the subcutaneous tumor tissue of mice. The release of six compounds had the potential of slow and controlled release, and could be well targeted to the stomach of mice. Therefore, potential salt curcumin derivatives are ideal potential anti-tumor prodrugs, and the resulting nanoparticles are suitable for delivery in organisms.