Colorectal cancer tissue-originated spheroids reveal tumor intrinsic signaling pathways and mimic patient clinical chemotherapeutic response as a rapid and valid model.

Locally advanced colorectal cancer requires preoperative chemotherapy to reduce local recurrence and metastasis rates, but it remains difficult to predict the tumor will be sensitive to which treatments. The patient-derived organoids (PDOs) are considered an effective platform for predicting tumor drug responses in precision oncology. However, it has the limitation of being time-consuming in practical applications, especially in neoadjuvant treatment. Here we used cancer tissue-originated spheroids (CTOS) method to establish organoids from a heterogeneous population of colorectal cancer specimens, and evaluated the capacity of CTOS to predict clinical drug responses. By analyzing the relationship of the activities of drug-treated CTOS, drug targets and target-related pathways, tumor intrinsic effective-target-related pathways can be identified. These pathways were highly matched to the abnormal pathways indicated by whole-exome sequencing. Based on this, we used half effective concentration gradients to classify CTOS as sensitive or resistant to chemotherapy regimens within a week, for predicting neoadjuvant treatment outcomes for colorectal cancer patients. The drug sensitivity test results are highly matched to the clinical responses to treatment in individual patients. Thus, our data suggested that CTOS models can be effectively screened ex vivo to identify pathways sensitive to chemotherapies. These data also supported organoid research for personalized clinical medication guidance immediately after diagnosis in patients with advanced colorectal cancer.

Quantitative dynamic contrast-enhanced parameters and intravoxel incoherent motion facilitate the prediction of TP53 status and risk stratification of early-stage endometrial carcinoma.

BACKGROUND: The aim of the study was to investigate the value of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and intravoxel incoherent motion (IVIM) in differentiating TP53-mutant from wild type, low-risk from non-low-risk early-stage endometrial carcinoma (EC). PATIENTS AND METHODS: A total of 74 EC patients underwent pelvic MRI. Parameters volume transfer constant (Ktrans), rate transfer constant (Kep), the volume of extravascular extracellular space per unit volume of tissue (Ve), true diffusion coefficient (D), pseudo-diffusion coefficient (D*), and microvascular volume fraction (f) were compared. The combination of parameters was investigated by logistic regression and evaluated by bootstrap (1000 samples), receiver operating characteristic (ROC) curves, calibration curves, and decision curve analysis (DCA). RESULTS: In the TP53-mutant group, Ktrans and Kep were higher and D was lower than in the TP53-wild group; Ktrans, Ve, f, and D were lower in the non-low-risk group than in the low-risk group (all P < 0.05). In the identification of TP53-mutant and TP53-wild early-stage EC, Ktrans and D were independent predictors, and the combination of them had an optimal diagnostic efficacy (AUC, 0.867; sensitivity, 92.00%; specificity, 80.95%), which was significantly better than D (Z = 2.169, P = 0.030) and Ktrans (Z = 2.572, P = 0.010). In the identification of low-risk and non-low-risk early-stage EC, Ktrans, Ve, and f were independent predictors, and the combination of them had an optimal diagnostic efficacy (AUC, 0.947; sensitivity, 83.33%; specificity, 93.18%), which was significantly better than D (Z = 3.113, P = 0.002), f (Z = 4.317, P < 0.001), Ktrans (Z = 2.713, P = 0.007), and Ve (Z = 3.175, P = 0.002). The calibration curves showed that the above two combinations of independent predictors, both have good consistency, and DCA showed that these combinations were reliable clinical prediction tools. CONCLUSIONS: Both DCE-MRI and IVIM facilitate the prediction of TP53 status and risk stratification in early-stage EC. Compare with each single parameter, the combination of independent predictors provided better predictive power and may serve as a superior imaging marker.

High ligation versus low ligation of the inferior mesenteric artery in laparoscopic rectal cancer surgery: a retrospective study on surgical and long-term outcome.

BACKGROUND: In laparoscopic low anterior resection for rectal cancer surgery, there has been controversy to whether the inferior mesenteric artery (IMA) should be ligated at the origin of its aorta (high ligation (HL)) or below the branches of the left colonic artery (LCA) (low ligation (LL)). This study was intended to clarify oncological outcome and long-term prognosis of retrospective analysis. METHODS: Analyzed the cases who underwent laparoscopic low anterior resection (LAR) in Shanghai Ruijin Hospital from January 2015 to December 2016, 357patients scheduled into 2 groups according to the level of IMA ligation: HL (n = 247) versus LL (n = 110). RESULTS: The primary endpoint is long-term outcomes, and the secondary endpoint is the incidence rate of major postoperative complications. There were no significant differences in 5-year overall survival (P = 0.92) and 5-year disease-free survival (P = 0.41). There were no differences between the clinical baseline levels in each group. The incidence of low anterior resection syndrome (LARS) in the two groups was statistically significant (P = 0.037). No significant differences were observed in operative time (P = 0.092) and intraoperative blood loss (P = 0.118). In the HL group, 6 cases (2.4%) had additional colonic excision due to poor anastomotic blood supply; none of the colonic anastomosis in the low ligation group had ischemic manifestations, and length from the proximal margin (P = 0.076), length from the distal margin (P = 0.184), the total number of lymph nodes excised (P = 0.065), and anastomotic leakage incidence (P = 0.33). CONCLUSION: Low ligation of the IMA which reserved LCA with vascular root lymph node dissection in laparoscopic low anterior resection for rectal cancer surgery may help protect the blood supply of the anastomosis, and will not increase postoperative complications while enhance recovery, without compromising radical excision and long-term prognosis.

Use of biexponential and stretched exponential models of intravoxel incoherent motion and dynamic contrast-enhanced magnetic resonance imaging to assess the proliferation of endometrial carcinoma.

Background: It is important to assess the proliferation of endometrial carcinoma (EC) noninvasively using imaging methods. This prospective diagnostic study investigated the value of biexponential and stretched exponential models of intravoxel incoherent motion (IVIM) and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in predicting the Ki-67 status of EC. Methods: In all, 70 patients with EC underwent pelvic MRI. The diffusion coefficient (D), pseudo diffusion coefficient (D*), perfusion fraction (f), distributed diffusion coefficient (DDC), water molecular diffusion heterogeneity index (α), volume transfer constant (Ktrans), rate transfer constant (Kep), and volume of extravascular extracellular space per unit volume of tissue (Ve) were compared. The area under the receiver operating characteristic (ROC) curve (AUC) was used to quantify diagnostic efficacy. Multivariate logistic regression and bootstrap (1,000 samples) analyses were used to establish and evaluate, respectively, the optimal model to predict Ki-67 status. Results: D, Ktrans, and Kep were lower while α was higher in the high-proliferation group as compared with low-proliferation group (all P values<0.05). D and Kep were independent predictors of Ki-67 status in EC, and the combination of these parameters had optimal diagnostic efficacy (AUC =0.920; sensitivity 85.71%; specificity 89.29%), which was significantly better than that of D (AUC =0.753; Z=2.874; P=0.004), α (AUC =0.715; Z=3.505; P=0.001), Ktrans (AUC =0.808; Z=2.741; P=0.006), and Kep (AUC =0.832; Z=2.147; P=0.032) alone. The validation model showed good accuracy (AUC =0.882; 95% confidence interval 0.861-0.897) and consistency (C-statistic =0.902). D, Kep, Ktrans, and α showed a slightly negative (r=-0.271), moderately negative (r=-0.534), slightly negative (r=-0.409), and slightly positive (r=0.488) correlation with the Ki-67 index, respectively (all P values <0.05). Conclusions: IVIM- and DCE-MRI-derived parameters, including D, α, Ktrans, and Kep, were associated with Ki-67 status in EC, and the combination of D and Kep may serve as a superior imaging marker for the identification of low- and high-proliferation EC.

Dysbiosis of human tumor microbiome and aberrant residence of Actinomyces in tumor-associated fibroblasts in young-onset colorectal cancer.

Colorectal cancer (CRC) is the third most common form of cancer, and the incidence of sporadic young-onset colorectal cancer (yCRC) has been increasing. Microbiota residing in the tumor microenvironment are emerging tumor components. The colonic microbiome differs between patients with CRC and healthy controls; however, few studies have investigated the role of the tumor microbiota in disease diagnosis and tumorigenesis of yCRC. We performed 16S rRNA sequencing analysis to identify the microbiome in CRC and found that tumor microbial diversity decreased in yCRC. Proteobacteria and Firmicutes were the most abundant phyla in all CRC samples, and Actinomyces and Schaalia cardiffensis were the key microbiota in the yCRC group. Correlation analysis revealed that Actinomyces co-occurred with various pro-tumor microbial taxa, including Bacteroidia, Gammaproteobacteria, and Pseudomonas. An independent cohort was used to validate the results. The Actinomyces in CRC was co-localized with cancer-associated fibroblasts and activated the TLR2/NF-κB pathway and reduces CD8+ T lymphocyte infiltration in CRC microenvironment. This study suggests that tumoral microbiota plays an important role in promoting tumorigenesis and therefore has potential as a promising non-invasive tool and intervention target for anti-tumor therapy.

CCL7 and TGF-ß secreted by MSCs play opposite roles in regulating CRC metastasis in a KLF5/CXCL5-dependent manner.

CXCL5 is overexpressed in colorectal cancer (CRC) and promotes distant metastasis and angiogenesis of tumors; however, the underlying mechanism that mediates CXCL5 overexpression in CRC remains unclear. Here, we successfully extracted and identified primary mesenchymal stromal cells (MSCs) and verified the promoting effects of tumor-associated MSCs on CRC proliferation and metastasis in vivo and in vitro. We found that MSCs not only promoted the expression of CXCL5 by secreting CCL7 but also secreted TGF-ß to inhibit this process. After secretion, CCL7/CCR1 activated downstream CBP/P300 to acetylate KLF5 to promote CXCL5 transcription, while TGF-ß reversed the effect of KLF5 on transcription activation by regulating SMAD4. Taken together, our results indicate that MSCs in the tumor microenvironment promoted the progression and metastasis of CRC and regulated the expression of CXCL5 in CRC cells by secreting CCL7 and TGF-ß. KLF5 is the key site of these processes and plays a dual role in CXCL5 regulation. MSCs and their secreted factors may serve as potential therapeutic targets in the tumor environment.

Research on ribosome-inactivating proteins from angiospermae to gymnospermae and cryptogamia.

Ribosome-inactivating Proteins (RIPs) are a group of cytotoxin proteins that usually contain a RNA N-glycosidase domain, which irreversibly inactivates ribosome, thus inhibiting protein synthesis. During the past 14 years (1990-2004), the studies conducted in our laboratory had been focusing on the structure and enzymatic mechanism of several PIPs. Herein, we briefly described a summary of the studies conducted mainly in our laboratory on RIPs from angiospermae to gymnospermae and cryptogamia as follows. (1) Cinnamomin is a novel type II RIP isolated from mature seeds of camphor tree. Like ricin, it specifically removes the adenine at A4324 in rat liver 28S rRNA. We systematically studied this low-toxic RIP in term of its enzymatic mechanism, the primary and crystal structure and the nucleotide sequence of its gene, the genetic expression, and its physiological role in the seed cell and the toxicity to human cancer cells and insect larvae. The cleavage of supercoiled double-stranded DNA was its intrinsic property of cinnamomin A-chain, its N- and C-terminal regions were found to be required for deadenylation of rRNA and also necessary for deadenylation of supercoiled double-stranded circular DNA. These results strongly excluded the possibility that cleavage of supercoiled DNA was due to nuclease contamination. (2) Trichosanthin, an abortifacient protein, was purified from the Chinese medicinal herb, Tian-hua-fen, obtained from root tubers of Chinese trichosanthes plant. We proved that trichosanthin was a RNA N-glycosidase, inactivating eukaryotic ribosome by hydrolyzing the N-C glycosidic bond of the adenose at site 4324 in rat 28S rRNA, and inhibited protein synthesis in vitro. (3) A unique Biota orientalis RNase (RNase Bo) was extracted from the mature seeds of the cypress cypress tree (Oriental arborvita), which was gymnospermae plant. It cleaved only a specific phosphodiester bond between C4453 and A4454 of 28S RNA in rat ribosomes, producing a small RNA-fragment (S-fragment), thus inhibiting protein synthesis and belonging to RNase-like RIP, similar to α-sarcin, a special RIP. (4) Lamjapin, the first RIP purified from kelp, the marine cryptogamia algal plant, was shown to be the first single-chained RNA N-glycosidase from marine plant to date. It hydrolyzed rat ribosomal 28S RNA to produce meanly a rather smaller RNA, shorter than the diagnostic R-fragment under the restricted condition. The significance of existence of type I RIP in the lower marine algal plant was briefly discussed.

Low-density lipoprotein receptor-related protein 1 is an essential receptor for trichosanthin in 2 choriocarcinoma cell lines.

Type-I ribosome-inactivating protein-trichosanthin (TCS) exhibits selective cytotoxicity toward different types of cells. It is believed that the cytotoxicity results from the inhibition of ribosomes to decrease protein synthesis, thereby indicating that there are specific mechanisms for TCS entry into target cells to reach the ribosomes. Low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) is a large scavenger receptor that is responsible for the binding and endocytosis of diverse biological ligands on the cell surface. In this study, we demonstrated that 2 choriocarcinoma cell lines can significantly bind and internalize TCS. In contrast, Hela cell line displayed no obvious TCS binding and endocytosis. Furthermore LRP1 gene silencing in JAR and BeWo cell lines blocked TCS binding; TCS could also interact with LRP1.The results of our study established that LRP1 was a major receptor for phagocytosis of TCS in JAR and BeWo cell lines and might be the molecular basis of TCS abortificient and anti-choriocarcinoma activity.

The lower cytotoxicity of cinnamomin (a type II RIP) is due to its B-chain.

The cytotoxicity of intact cinnamomin (a type II ribosome-inactivating protein, RIP) and the RNA N-glycosidase activity of cinnamomin A-chain have been studied and compared with those of ricin. Cinnamomin A-chain exhibits a similar RNA N-glycosidase activity in inhibiting in vitro protein synthesis compared with that of ricin, whereas the cytotoxicity to BA/F3beta cells of intact cinnamomin is markedly lower than intact ricin. In order to demonstrate that it is the B-chains of the two RIPs that bear the difference in cytotoxicity, two hybrid RIPs are prepared from the purified A-/B-chains of cinnamomin and ricin by the disulfide exchange reaction. It has been found that hybrid RIP constructed from cinnamomin A-chain and ricin B-chain is more toxic to BA/F3beta cells than the native cinnamomin, and equivalent to the native ricin. However, the cytotoxicity to BA/F3beta cells of the hybrid RIP constructed from the ricin A-chain and cinnamomin B-chain is lower than ricin, equivalent to the native cinnamomin. Furthermore, the bound amounts of two B-chains on the cell surface are determined by the method of direct cellular ELISA and Scatchard analysis of the binding of the two B-chains indicates that cinnamomin and ricin share similar binding sites with different affinity.

The three-dimensional structure and X-ray sequence reveal that trichomaglin is a novel S-like ribonuclease.

Trichomaglin is a protein isolated from root tuber of the plant Maganlin (Trichosanthes Lepiniate, Cucurbitaceae). The crystal structure of trichomaglin has been determined by multiple-isomorphous replacement and refined at 2.2 A resolution. The X-ray sequence was established, based on electron density combined with the experimentally determined N-terminal sequence, and the sequence information derived from mass spectroscopic analysis. X-ray sequence-based homolog search and the three-dimensional structure reveal that trichomaglin is a novel S-like RNase, which was confirmed by biological assay. Trichomaglin molecule contains an additional beta sheet in the HV(b) region, compared with the known plant RNase structures. Fourteen cystein residues form seven disulfide bridges, more than those in the other known structures of S- and S-like RNases. His43 and His105 are expected to be the catalytic acid and base, respectively. Four hydrosulfate ions are bound in the active site pocket, three of them mimicking the substrate binding sites.

Cinnamomin--a versatile type II ribosome-inactivating protein.

Ribosome-inactivating proteins (RIPs) are a group of toxic proteins that can specifically act on the universally conserved sarcin/ricin domain (S/R domain) of the largest RNA in ribosome and thus irreversibly inactivate ribosome for protein synthesis. Cinnamomin is a multifunctional type II RIP isolated in our laboratory from the mature seeds of the camphor tree. This protein has been extensively studied with regard to its purification, characteristics, structure and function, genetic expression, enzymatic mechanism, physiological role in seed cell and toxicity to cancer cells and insect larvae. The research results of cinnamomin obtained in our laboratory are summarized in this review. Understanding of cinnamomin and the relative new proteins will help expand our knowledge of RIPs and may accelerate theoretical study and the development of their potential applications.

Enzymatic activity of toxic and non-toxic type 2 ribosome-inactivating proteins.

Ribosome-inactivating proteins (RIPs) display adenine polynucleotide glycosylase activity on different nucleic acid substrates, which at the ribosomal level is responsible for the arrest of protein synthesis. Some type 2 RIPs, namely ricin and related proteins, are extremely toxic to mammalian cells and animals whilst other type 2 RIPs (non-toxic type 2 RIPs) display three to four logs less toxicity. We studied whether a correlation exists between toxicity on cells and enzymatic activity on nucleic acids. All type 2 RIPs differ in their depurinating activity on the different substrates with differences of up to one to two logs. The toxicity of type 2 RIPs is independent of their enzymatic activity on nucleic acids or on ribosomes.

Both N- and C-terminal regions are essential for cinnamomin A-chain to deadenylate ribosomal RNA and supercoiled double-stranded DNA.

Cinnamomin is a type II ribosome-inactivating protein and its A-chain exhibits RNA N-glycosidase activity to remove an adenine in the conserved sarcin/ricin loop of the largest RNA in ribosome, arresting protein synthesis at the elongation step. In this report, deadenylation of both rRNA and supercoiled DNA by native and recombinant cinnamomin A-chain expressed in Escherichia coli was demonstrated. However, the mutants of cinnamomin A-chain devoid of N-terminal 52 or/and C-terminal 51 amino acid residues lost both the activity of RNA N-glycosidase and the ability to release adenines from supercoiled DNA. Additionally, supercoiled DNA could not be cleaved into nicked and linear forms by these mutants. These results indicate that both N- and C-terminal regions are essential for the cinnamomin A-chain to deadenylate rRNA and supercoiled DNA. It was suggested that phosphodiester bonds in the extensively deadenylated region of supercoiled DNA would become fragile and liable to be broken spontaneously owing to the existence of tension in the supercoiled DNA.

The role of tyrosine residues in the RNA N-glycosidase activity of cinnamomin A-chain.

Cinnamomin is a type II ribosome-inactivating protein (RIP) and its A-chain (CTA) is a RNA N-glycosidase. It is observed that modification of tyrosine residues by N-acetylimidazole (N-AI) causes almost complete loss of CTA activity. Adenine partially protects tyrosine residues from modification by N-AI. It is proposed that tyrosine residues are involved in the active site of CTA and they are crucial in recognition and binding of ribosomal RNA. Tryptophan residues of CTA are also studied by NBS modification.

Cinnamomin: a multifunctional type II ribosome-inactivating protein.

Plant ribosome-inactivating proteins (RIPs) are a group of toxic proteins that can irreversibly inactivate ribosomes by specifically removing the conserved adenine base from the "Sarcin/Ricin domain" of the 28S RNA in ribosome. Cinnamomin is a novel type II RIP isolated in our laboratory from the mature seeds of camphor tree. Besides site-specific deadenylation of the A4324 in the Sarcin/Ricin domain of rat ribosome, this protein could also release the adenine base from DNA molecules at multiple sites and from AMP, ADP, dAMP and adenosine. Furthermore, cinnamomin displays cytotoxicity to carcinoma cells and insect larvae by modifying their ribosomal RNA. These functions possessed by cinnamomin shed a new light on the possible application of cinnamomin in the field of immunotoxin design and transgenic reagents. In this review, we introduce the major recent results on cinnamomin obtained in our laboratory, including purification of this protein, characterization of its enzymatic mechanism, structure and function, gene pattern, physiological role and its biological implications in cytotoxicity.

Low content of protein S29 in ribosomes of human lung cancer cell line a549: detected by two-dimensional electrophoresis.

This study revealed that the content of protein S29 in ribosomes of cancer cell line A549 was distinctly low (equivalent to about 30% of that of 2BS). The conclusion was acquired based on the ratios of spot volume of ribosomal protein S29 to that of several other ribosomal proteins (S29/L37a, S29/L38, S29/S27 and S29/S28) in the same gel plate. The possible biological roles of ribosomal protein S29 in malignant transformation and translation regulation are briefly discussed.

Isolation and enzymatic characterization of lamjapin, the first ribosome-inactivating protein from cryptogamic algal plant (Laminaria japonica A).

Lamjapin, a novel type Iota ribosome-inactivating protein, has been isolated from kelp (Laminaria japonica A), a marine alga. This protein has been extensively purified through multiple chromatography columns. With a molecular mass of approximately 36 kDa, lamjapin is slightly larger than the other known single-chain ribosome-inactivating proteins from the higher plants. Lamjapin can inhibit protein synthesis in rabbit reticulocyte lysate with an IC50 of 0.69 nm. It can depurinate at multiple sites of RNA in rat ribosome and produce the diagnostic R-fragment and three additional larger fragments after the aniline reaction. Lamjapin can deadenylate specifically at the site A20 of the synthetic oligoribonucleotide (35-mer) substrate that mimics the sarcin/ricin domain (SRD) of rat ribosomal 28S RNA. However, it cannot hydrolyze the N-C glycosidic bond of guanosine, cytidine or uridine at the corresponding site of the A20 of three mutant SRD RNAs. Lamjapin exhibits the same base and position requirement as the ribosome-inactivating proteins from higher plants. We conclude that lamjapin is an RNA N-glycosidase that belongs to the ribosome-inactivating protein family. This study reports for the first time that ribosome-inactivating protein exists in the lower cryptogamic algal plant.

Nonspecific deadenylation on sarcin/ricin domain RNA catalyzed by gelonin under acidic conditions.

Gelonin is a single-chain ribosome-inactivating protein that can hydrolyze the glycosidic bond of a highly conserved adenosine residue in the sarcin/ricin domain (SRD) of the largest RNA in ribosome and thus irreversibly inhibit protein synthesis. Recently, the specificity in substrate recognition was challenged by the fact that gelonin could remove adenines from some other oligoribonucleotide substrates. However, the site specificity of gelonin to deadenylate various substrates were unknown. Hereby, the effect of pH values upon site specificity of the deadenylation activity of gelonin was studied using the synthetic oligoribonucleotide (named SRD RNA) that mimicked the ribosomal SRD. Interestingly, gelonin gradually acquired the ability to nonspecifically remove adenines from SRD RNA when pH values changed from neutral to acidic conditions. Another two SRD RNA mutants, either with the conserved adenosine deleted or with the tetraloop converted, showed very similar cleavage style to wild-type SRD RNA, underscoring the important role of pH value in site specificity of recognition by gelonin. Furthermore, the RNA N-glycosidase activity of gelonin was also enhanced with the decreasing of pH values. In addition, no obvious change was observed in the molecular conformation of gelonin at various pH values. Taken together, our data implied that the protonation of adenosines in SRD RNA was potentially an important factor for the nonspecific deadenlyation by gelonin.