[Application of the two-step sentinel lymph node biopsy with double-tracer in early staged endometrial cancer].

Objective: To explore the feasibility and clinical value of sentinel lymph node (SLN) biopsy through cervix-uterine combined two-step injection with two tracers in patients with early stage endometrial cancer. Methods: From July 2019 to April 2021, a total of 73 patients, aged (54.2±3.3) year, who were preoperatively diagnosed as stage Ⅰ-Ⅱ endometrial cancer (including 56 low-risk patients and 17 medium-high risk patients) in Affiliated Hospital of Qingdao University were selected. According to the different sites of tracer injection, the patients were randomly divided into three groups: cervical injection group (25 cases): 1 ml of nano-carbon was used to inject at 3 and 9 o'clock in the cervix; uterine injection group (21 cases): the magnetic resonance imaging examination was performed to determine the location of the lesion, and 4 ml of methylene blue was injected into the uterine body at 2 sites where the lesion was located; combined injection group (27 cases): cervical injection of nano-carbon (1 ml) combined with uterine injection of methylene blue (4 ml). The SLN in all patients were identified under laparoscopy, removed, and followed by frozen pathological examination. Pathological ultra-staging was performed if the postoperative pathological outcome of SLN was negative. The total detection rate of SLN, bilateral pelvic SLN detection rate, sensitivity, negative predictive value, and location of SLN in each group were calculated and compared. Results: (1) In 73 patients with endometrial cancer, the overall detection rate of SLN was 88% (64/73), the detection rate of bilateral pelvic SLN was 67% (49/73), and the detection rate of para-aortic SLN was 49% (36/73). The overall detection rate of SLN (71%, 15/21) and bilateral pelvic SLN (43%, 9/21) in the intrauterine injection group were significantly lower than those in the cervical injection group [92% (23/25), 76% (19/25), respectively] and the combined injection group [96% (26/27), 78% (21/27), respectively; all P<0.05]; the detection rate of para-aortic SLN in the cervical injection group (28%, 7/25) was significantly lower than those in the intrauterine injection group and combined injection group [52% (11/21) and 67% (18/27), respectively; both P<0.05]. Among 73 cases with endometrial cancer, 9 had lymph node metastasis confirmed by postoperative pathological examination, 8 of them had lymph node metastasis detected by SLN and 1 had no lymph node metastasis detected by SLN, with a total sensitivity of 89% and a negative predictive value of 98%. The sensitivity and negative predictive value of cervical injection group and combined injection group were 100%, while the sensitivity and negative predictive value of intrauterine injection group were 67% and 95%. Among 56 low-risk patients, only one patient with lymph node metastasis was confirmed by postoperative pathology by SLN detection, and the metastasis rate was 2% (1/56), and the sensitivity and negative predictive value were 100%. Lymph node metastasis was confirmed in 8 of 17 patients (8/17) with a sensitivity of 88% and a negative predictive value of 90%. (2) A total of 459 SLN were detected in 73 endometrial cancer patients, with the highest proportion of external iliac (33.3%, 153/459).The obturator foramen was 25.3% (116/459), para-aortic 19.6% (90/459), iliac 12.0% (55/459), and presacral 9.8% (45/459). The proportion of para-aortic SLN in the cervical injection group was 12.4% (21/169), which were significantly lower than that in the intrauterine injection group and the combined injection group [27.4% (26/95) and 22.1% (43/195), respectively; both P<0.05]. (3) Pathological super-staging results: among 64 patients with negative SLN routine paraffin pathology, 4 cases of lymph node micro-metastases and 1 case of isolated tumor cell metastasis were detected, and the SLN micro-metastases rate was 8% (5/64), including 2 cases of low-risk patients and 3 cases of medium-high risk patients. Conclusions: SLN biopsy has high sensitivity and negative predictive value in patients with early endometrial cancer and could be used as an alternative to systematic lymph node dissection in low-risk patients. The SLN mapping through cervical-uterine combined injection could further improve the detection rate effectively and avoid the missed detection of positive para-aortic lymph node, especially for high-risk patients or patients with fundal tumor involvement.

[Effects of ascites derived exosomes on the stemness and invasion ability of ovarian cancer stem-like cell].

Objective: To investigate the effects of ovarian cancer ascites-derived exosomes on the stem cell properties and invasion ability of ovarian cancer stem-like cell (OCS-LC). Methods: (1) A2780 cells were induced into OCS-LC in serum-free medium, and authenticating their stem-like properties by sphere-forming test, differentiation test and CD133 marker detection. (2) Exosomes from ascites and A2780 cell were extracted by ultracentrifugation, then authenticating them. The morphology of exosomes was observed by the transmission electron microscope, exosome particle size was measured by nanoparticle tracking analysis (NTA). The expressions of heat shock protein 70 (HSP-70), CD63 and CD9 were detected by western blot. (3) The exosomes from ovarian cancer ascites and tumor cell supernate were co-cultured with OCS-LC. The groups were divided into control group, ascites-derived exosomes (ADE) group (ADE+OCS-LC group), and cells-derived exosomes (CDE) group (CDE+OCS-LC group). The sphere-forming ability was evaluated by sphere-forming cycle, maximum sphere diameter and sphere-forming rate of each group; the expression of CD133 was detected by immunofluorescence staining under microscope; quantitative real-time (qRT)-PCR was used to detected the expression levels of octamer-4 (Oct-4), Nanog mRNA of the signature genes in the stem cells of each group; the metastasis ability of each group was measured by transwell assay. Results: (1) Identification of OCS-LC: sphere-forming experiment showed that the suspension of OCSC single cells was grown in serum-free medium in secondary sphere-forming. Differentiation function experiment showed that OCS-LC were differentiated into adherent A2780 cells by changing the growth mode in serum containing medium. Flow cytometry showed that the proportion of CD133 positive (CD133+) cells in OCS-LC group was (18.9±0.9)%, significantly higher than that of control group (0.6±0.5)% (t=38.570, P<0.01). (2) Under transmission electron microscope, clear lipid bilayer structure was observed in ADE and CDE, and one side presented a concave hemispheric or cup like structure. NTA showed that the diameter of exosomes mainly ranged from 30 to 100 nm, with an average diameter of 67.2 nm. Western blot analysis showed that the specific protein molecules HSP-70, CD63 and CD9 were positive. (3) Three groups' OCS-LC could continue to grow into spheres, and the group of ADE+OCS-LC showed two growth modes, most of the cells continued to grow into spheres, while a small part of cells grew in adherent differentiation. The sphere-forming rate of OCS-LC in the control group, ADE+OCS-LC group, and CDE+OCS-LC group were (1.05±0.20)%, (4.15±0.10)%, and (10.45±0.25)%, the sphere-forming cycle of OCS-LC in the three groups were (15.3±1.5), (10.3±0.6), and (6.7±0.6) days, and the maximum diameters of OCS-LC were (100.3±3.2), (145.2±5.1) and (170.0±2.1) µm, respectively. And the differences were statistically significant (all P<0.05). After co-culture of exosomes with OCS-LC, the sphere-forming ability of cells in the group of CDE+OCS-LC was prior to ADE+OCS-LC group (all P<0.05). Immunofluorescence staining showed that the number of CD133 green fluorescent chromophore cells in OCS-LC groups [(46.2±2.1)%, (58.4±2.2)%] was significantly higher than that in the control group [(26.6±1.5)%] after the addition of exosomes in co-culture, the positive rate of CD133 was higher than that in the control group(F=187.588, P<0.05). The qRT-PCR results showed that the expression levels of Oct-4 mRNA in ADE+OCS-LC and CDE+OCS-LC groups were 3.46±0.24, 4.03±0.31, compared with that in control group (1.04±0.12), the differences were statistically significant (F=134.932, P<0.05). The mRNA expression levels of Nanog were 1.57±0.32, 2.66±0.15, which were significantly higher than that in the control group (1.00±0.07), and the differences were statistically significant (F=49.329, P<0.05). And the expression of both in CDE+OCS-LC group increased more significantly than ADE+OCS-LC group (all P<0.05). The number of invasive cells in the three groups of OCS-LC were: control group 30±5, ADE+OCS-LC group 102±4, CDE+OCS-LC group 210±7, and there were statistically significant differences among three groups (F=820.800, P<0.05). Compared with the control group, the number of invaded cells in the co-culture group were significantly increased (P<0.05), and the CDE+OCS-LC group had the higher cell invasion ability then the ADE+OCS-LC group (t=23.202, P<0.05). Conclusions: Exosomes derived from ovarian cancer ascites could enhance and maintain the stemness of OSC-LC, and promote the invasion of tumor cells. Moreover, CDE is superior to ADE.

[Expression and function of long intergenic non-protein coding RNA-regulator of reprogramming in high-grade ovarian serous cancer].

Objective: To investigate the expression of long intergenic non-protein coding RNA-regulator of reprogramming (Linc-ROR) in high-grade ovarian serous cancer, and explore the relationship between Linc-ROR expression and biological function of high-grade ovarian serous cancer. Methods: A total of 34 high-grade ovarian serous cancer tissue samples and 19 normal fallopian tube tissue samples were collected between June 2014 and February 2016. Real-time reverse transcription (RT)-PCR was used to detect the Linc-ROR mRNA expression in different samples. The relationship between Linc-ROR expression level and ovarian cancer International Federation of Gynecology and Obstetrics (FIGO) stage, lymph node metastasis was analyzed. Constructed Linc-ROR small interference RNA (siRNA) and pIRES2-EGFP-Linc-ROR plasmid, then Linc-ROR siRNA and pIRES2-EGFP-Linc-ROR plasmid were respectively transfected into SKOV3 cells. Cell proliferation, migration and invasion ability were assessed by cell counting kit-8 (CCK-8), wound healing assay and transwell invasion assay. Results: (1) The expression level of Linc-ROR mRNA was significantly higher in high-grade ovarian serous cancer than normal fallopian tube tissues (4.31± 0.38 vs 1.03 ± 0.21; t=25.842, P<0.01). With the progression of FIGO stages, the expression of Linc-ROR was increased (F=95.702, P<0.01), and it was associated with lymph node metastasis (t=7.397, P<0.01). (2) The results of RT-PCR showed that the expression level of linc-ROR in Linc-ROR-i group was significantly lower than that in Linc-ROR-NC-i group (0.30 ± 0.11 vs 1.02 ± 0.10; t=15.269, P<0.01). The expression level in Linc-ROR-p group was significantly higher than that in Linc-ROR-NC-p group (8.90± 0.45 vs 1.03±0.17; t=21.934, P<0.01). The CCK-8 assay showed that when the cells were cultured for 3, 4, 5 and 6 days, the A value in Linc-ROR-i group was significantly lower than that in Linc-ROR-NC-i group (P< 0.05). And the A value in Linc-ROR-p group was significantly higher than that in Linc-ROR-NC-p group (P< 0.05). Wound healing assay showed that, after 48 hours incubation, migration rate of cells in Linc-ROR-i group was significantly less than that in the Linc-ROR-NC-i group [(52±4)% vs (67±5)%; t=5.720, P< 0.01]. The migration of cells in Linc-ROR-p group was significantly greater than that in the Linc-ROR-NC-p group [(84±4)% vs (66±4)%; t=7.330, P <0.01]. Cell transwell invasion assay showed that, after 48 hours of incubation, the number of invasive cells in Linc-ROR-i group was lower than that in Linc-ROR-NC-i group (74 ± 3 vs 104 ± 3; t=15.810, P<0.01). And the number of invasive cells in Linc-ROR-p group was higher than that in Linc-ROR-NC-p group (217 ± 4 vs 108 ± 5; t=38.060, P<0.01). Conclusion: Highly expressed Linc-ROR could enhance the proliferation, migration and invasion ability of high-grade ovarian serous cancer cells, which may be one of the important molecules in the occurrence and development, invasion and metastasis of high-grade ovarian serous cancer.

A contraceptive peptide vaccine targeting sulfated glycoprotein ZP2 of the mouse zona pellucida.

In this study, we have mapped and characterized a B cell epitope of sulfated glycoprotein ZP2 (ZP2) as a step toward the development of a multi-epitope zona pellucida (ZP) vaccine. Recombinant polypeptides expressed by random deoxyribonuclease-digested fragments of ZP2 cDNA were screened for binding to IE-3, a monoclonal antibody to murine ZP2. Positive clones contained cDNA inserts encoding polypeptide corresponding to ZP2(103-134). When normal or ovariectomized female mice were immunized with three overlapping peptides that span this region of ZP2 (101-120, 111-130, 121-140), only ZP2(121-140) elicited IgG antibodies that reacted with mouse ovarian ZP, indicative of the presence of native B epitope and helper T cell epitope in ZP2(121-140). To more finely map the ZP2 B cell epitope, a random peptide display library was screened with the IE-3 antibody, and a consensus tetramer sequence VxYK that matched the ZP2(123-126) sequence VRYK was located. Competitive immunofluorescence analysis with single alanine-substituted VxYK peptides ranked the relative contribution of the three critical B cell epitope residues as Y > V > K. A chimeric peptide was constructed that contained the YRYK motif of ZP2 and a bovine RNase T cell epitope. Although (C57BL/6xA/J) F1 (B6AF1) female mice immunized with the chimeric peptide developed ZP antibody response, this peptide elicited antibody only in mice of the histocompatibility complex (MHC) H-2(k or b) haplotype. In contrast, ZP2(121-140) peptide elicited antibody in inbred mice with three additional mouse MHC haplotypes. Moreover, although ZP2(121-140) contained a T cell epitope, no oophoritis was observed after immunization of B6AF1 mice with ZP2(121-140) in complete Freund's adjuvant (CFA). In a preliminary trial, female B6AF1 mice immunized with ZP2(121-140) in CFA had reduced litter sizes as compared with mice injected with CFA alone.

Antibody responses and infertility in mice following oral immunization with attenuated Salmonella typhimurium expressing recombinant murine ZP3.

Ovarian ZP3, the primary sperm receptor, is a major glycoprotein of mouse zona pellucida (ZP). Because antibodies raised against ZP3 block sperm-egg interaction, ZP3 has been considered a candidate immunogen in the development of a contraceptive vaccine. This study explored the possibility of using an attenuated Salmonella typhimurium vaccine strain expressing recombinant ZP3 to elicit an antibody response and infertility in mice. A cDNA sequence generated by the polymerase chain reaction encoding 342 amino acid residues (23-364) of the mouse (m)ZP3 was cloned into an Asd+ vector. An avirulent Salmonella vaccine strain stably expressed the ZP3 polypeptide and colonized the internal organs of mice after oral inoculation. Oral immunization of female BALB/c mice with the recombinant Salmonella vaccine strain expressing mZP3 induced significant levels of anti-native ZP IgG antibodies in serum and IgA antibodies in vaginal secretions. The IgG antibodies thus induced also bound to ZP in vivo. When mated with males, 3 of 6 females immunized with the recombinant Salmonella were infertile. In contrast, none of the mice that received Salmonella containing the vector plasmid produced antibodies to ZP and all were fertile. No ovarian inflammation was observed in the immunized mice at autopsy. The results suggest a potential oral contraceptive vaccine to control populations of rodent vectors of disease and to induce reversible infertility in humans.

Contraceptive vaccine assessment based on a murine ZP3 mini-autoantigen.

A summary is presented of published and some unpublished observations from studies on the immunological response of mice to a 13-mer peptide of the murine ovarian zona pellucida glycoprotein ZP3. The findings have the following implications for the design of immunocontraceptive vaccines. To be reversible, a ZP3 vaccine must not contain pathogenic T cell epitopes of ZP3, but contraception without autoimmune oophoritis may be feasible. The immune response to the ZP3 mini-autoantigen is highly variable among inbred mouse strains, suggesting that a single oophoritogenic peptide would not achieve irreversible contraception in an outbred population. The discovery of antigen mimicry at the level of T cell peptide has thrown doubt on the validity of current strategy in detecting relevant self-antigens that might cross react with vaccine immunogens and on the feasibility of fully predicting the cross-reactive autoimmunogenic potential of a peptide or polypeptide vaccine antigen. Autoantibodies directed against epitopes outside the ZP3 mini-autoantigen, produced by immunization with the pure T cell epitope, react with high affinity, with native zona pellucida, and may be useful in identifying B cell epitopes in ZP3.