Development Of Peritoneal Metastasis Research Articles

Vagus nerve signal has an inhibitory influence on the development of peritoneal metastasis in murine gastric cancer

The vagus nerve is the only pathway for transmitting parasympathetic signals between the brain and thoracoabdominal organs, thereby exhibiting anti-inflammatory functions through the cholinergic anti-inflammatory pathway. Despite often being resected during lymph node dissection in upper gastrointestinal cancer surgery, the impact of vagotomy on postoperative outcomes in gastric cancer patients remains unclear. Sub-diaphragmatic vagotomy was performed on C57BL/6 mice. Three weeks later, syngeneic murine gastric cancer cell line YTN16P was injected into the peritoneal cavity, and the number of peritoneal metastases (PM) on the mesentery and omentum compared with control mice. The phenotypes of immune cells in peritoneal lavage and omental milky spots one day after tumor inoculation were analyzed using flow cytometry and immunohistochemistry. Intraperitoneal transfer of 3 × 105 YTN16P significantly increased the number of metastatic nodules on the mesentery in the vagotomy group compared to the control group. The omental metastasis grade was also significantly higher in the vagotomy group. Phenotypic analysis of immune cells in peritoneal lavage did not reveal significant differences after vagotomy. However, vagotomized mice exhibited a notable increase in milky spot area, with a higher presence of cytokeratin(+) tumor cells, F4/80(+) macrophages, and CD3(+) T cells. Vagus nerve signaling appears to regulate the immune response dynamics within milky spots against disseminated tumor cells and inhibits the development of PM. Preserving the vagus nerve may offer advantages in advanced gastric cancer surgery to reduce peritoneal recurrence.

The impact of body weight on the development of peritoneal metastases in colorectal cancer patients: results from a nationwide cohort study

BackgroundObesity is a major global health problem and an important risk factor for colorectal cancer (CRC) is increased body weight. Obesity plays a role in the peritoneal dissemination of cancer; however, it is unclear whether this also applies for peritoneal dissemination of CRC. The purpose of this study was to provide insight in the role of obesity on the peritoneal dissemination of colorectal cancer.MethodsOf all patients diagnosed with CRC in the Netherlands in the first half of 2015, follow-up data was completed in 2019. Weight at time of primary diagnosis was categorized as underweight, normal weight, overweight, or obese. Logistic regression modelling was used to assess the association between weight and the presence of synchronous colorectal peritoneal metastases (CPM), and Cox regression modelling was used to assess the association between weight and metachronous CPM. Patient and tumor characteristics were taken into account. The analyses were adjusted for tumor stage, nodal stage, tumor location, and tumor histology.ResultsIn total, 6436 patients were included in this study. Two-hundred ninety-three (4.6%) patients presented with synchronous CPM at the time of primary diagnosis, while another 278 (5.1%) patients developed metachronous CPM after a median time of 16.5 months. Univariable and multivariable logistic regression modelling did not identify an effect of weight on the presence of synchronous CPM. Neither underweight (odds ratio [OR] 1.10, 95% CI 0.48–2.54), nor overweight (OR 0.96, 95% CI 0.71–1.29), or obesity (OR 0.84, 95% CI 0.56–1.26) was either positively or negatively associated with the presence of synchronous peritoneal metastases as compared to normal weight. Univariable and multivariable Cox regression modelling did not identify an effect of weight on the development of metachronous CPM. Neither underweight (HR 0.162, 95% CI 0.02–1.16), nor overweight (HR 1.07, 95% CI 0.82–1.39), or obesity (HR 1.02, 95% CI 0.73–1.16) was either positively or negatively associated with the presence of synchronous peritoneal metastases as compared to normal weight.ConclusionCRC patients who are overweight or obese are not more at risk for the presence of synchronous CPM nor development of metachronous CPM than their normal-weight counterparts.

Abstract 3599: Molecular characterization of mouse colorectal cancer cell lines with high potential of peritoneal metastasis

Abstract Background: Peritoneal metastasis occurs in approximately 7% of colorectal cancer (CRC) patients and is associated with worse prognosis compared to non-peritoneal metastasis. In CRC patients with peritoneal metastasis, administration of systemic chemotherapy only slightly improves overall survival. Therefore, a better understanding of the biology of peritoneal metastasis and the development of new molecular therapeutics for CRC patients with peritoneal metastasis are urgently needed. Materials and Methods: CT26 mouse CRC cell lines with low metastatic potentials (CT26-N5) and high metastatic potentials to peritoneum (CT26-P6) were established after several rounds of in vivo selection by orthotopic transplantation of CT26 cells into the syngeneic BALB/c mice. Transcriptomic and proteomic analyses were conducted on CT26-N5 and CT26-P6 cells. Results: Orthotopic transplantation of CT26-P6 cells exhibited significantly increased peritoneal metastasis compared to CT26-N5 cells, while growth of primary tumors was not different between these CT26 sublines. In addition, CT26-P6 cells had higher cell migration and invasion potential in vitro than CT26-N5 cells. Integrated analyses of transcriptome and proteome resulted in identification of molecular signatures associated with peritoneal metastasis. An actin-binding protein Advillin, encoded by the Avil gene, was markedly increased at both mRNA and protein levels in CT26-P6 cells compared to CT26-N5 cells. Knockdown of Avil using siRNA or shRNA in CT26-P6 cells significantly reduced cell migration and invasion in vitro and occurrence of peritoneal metastasis in vivo. Gene Set Enrichment Analysis on transcriptome datasets from CT26-P6 cells and CT26-N5 cells identified interferon gamma response as the most significantly enriched pathway in CT26-cells. In addition, Immunoprecipitation coupled with LC-MS/MS identified potential interaction between Avil and a molecule X that regulates epithelial-mesenchymal transition in CRC. Consistent with the findings in mouse CT26 cells, AVIL knockdown in CRC patient-derived cells (PDCs) suppressed cell migration and invasion in vitro. While AVIL expression was induced by interferon gamma in CRC PDCs, JAK inhibitors, Momelotinib and Ruxolitinib, significantly reduced interferon gamma-induced AVIL expression, suggesting regulation of AVIL by interferon gamma. Conclusion: Molecular characterization of mouse colorectal cancer cell lines with different metastatic potential identified AVIL as a potential therapeutic target in CRC patients with peritoneal metastasis. Regulation of AVIL expression by interferon gamma may suggest functional relevance of tumor immune microenvironment in the development of peritoneal metastasis in CRC. Citation Format: Haruki Mori, Hisanori Isomura, Shuang Zhou, Taisuke Kajino, Yuichi Abe, Takashi Kinoshita, Seiji Natsume, Yusuke Sato, Akira Ouchi, Toru Miyake, Waki Hosoda, Koji Komori, Yasuhiro Shimizu, Masaji Tani, Ayumu Taguchi. Molecular characterization of mouse colorectal cancer cell lines with high potential of peritoneal metastasis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3599.

The utility of ctDNA in colorectal cancer with peritoneal metastases.

The development of peritoneal metastases (PM) in patients with colorectal cancer (CRC) connotates a poor prognosis. Circulating tumour (ctDNA) is a promising tumour biomarker in the management CRC. This systematic review aimed to summarize the role of ctDNA in patients with CRC and PM. Following the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines, a systematic review of the literature until June 2022 was performed. Studies reporting on the utility of ctDNA in colorectal PM were included. A total of eight eligible studies were identified including a total of 167 patients. The findings from this review suggest an evolving role for ctDNA in CRC with PM. ctDNA can be isolated from both plasma and peritoneal fluid, with peritoneal fluid preferred as the liquid biopsy of choice with higher mutation detection rates. Concordance rates between tissue and plasma/peritoneal ctDNA mutation detection can vary, but is generally high. ctDNA has a potential role in monitoring anti-EGFR treatment response and resistance, as well as in predicting future prognosis and recurrence. The detection of ctDNA in plasma of patients with isolated PM is also possibly suggestive of occult systemic disease, and patients exhibiting such ctDNA positivity may benefit from systemic treatment. Limitations to ctDNA mutation detection may include the size of peritoneal lesions, as well as the fact that PM poorly shed ctDNA. While these findings are promising, further large-scale studies are needed to better evaluate the utility of ctDNA in this subset of patients.

Abstract 6284: Exosomal miR-29b derived from mesenchymal stem cell (MSC) may suppress peritoneal metastasis via the effects on peritoneal mesothelial cells

Abstract Background: Mesothelial cells play tumor-promoting roles in the development of peritoneal metastasis (PM) through mesothelial-mesenchymal transition (MMT). We reported miR-29b suppressed MMT of human peritoneal mesothelial cell (HPMC). Methods: MiR-29b mimics and negative control miRNA (NC) were transfected to human bone marrow derived MSC by lipofection method. Exosomes were purified from the culture supernatants of the MSC with ultracentrifugation, and added to HPMC. Proliferation and migration of HPMC were examined with MTT assay and transwell assay, respectively. MMT of HPMC induced by 10 ng/ml TGF-β, and evaluated with immunofluorescence staining. Results: MSC transfected with miR-29b mimics produced significantly increased amounts of miR-29b-containing exosomes (p<0.0001, n=3) as compared with negative control. After 24h-culture with PKH-26 stained exosomes, HPMC actively took the exosomes. After 48 hr-culture with TGF-β, HPMC apparently changed the morphology from round to spindle shape. The expression level of E-cadherin and Calretinin in HPMC was decreased while vimentin and Fibronectin tended to be upregulated by TGF-β. However, exosomes from miR-29b transfected MSC significantly suppressed the morphological changes and reduced the expression levels of vimentin and Fibronectin with restored expression of E-cadherin and Calretinin, suggesting the inhibition of MMT in HPMC. The exosome inhibited the proliferation and migration of HPMC by 31±14% (p<0.01, n=5) and 67±9% (p<0.01, n=3), and inhibited the adhesion of NUGC by 90±3% (p<0.001, n=5) as compared with NC exosome. Conclusion: Exosomes from MSC transfected with miR-29b mimic efficiently suppresses MMT. Replacement of the miR-29b in peritoneal cavity might be used for the treatment of PM partially via the effects on mesothelial cell. Citation Format: Yuki Kimura, Hideyuki Ohzawa, Yuki Kaneko, Kohei Tamura, Yurie Futoh, Kazuya Takahashi, Akira Saito, Mineyuki Tojo, Hideyo Miyato, Joji Kitayama. Exosomal miR-29b derived from mesenchymal stem cell (MSC) may suppress peritoneal metastasis via the effects on peritoneal mesothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6284.

Association between biopsy method and development of peritoneal metastases in perihilar cholangiocarcinoma.

In patients with perihilar cholangiocarcinoma (PHC), there is concern that transperitoneal (TP) biopsy may seed tumor in the peritoneal cavity, increasing risk of peritoneal metastases (PM). A retrospective review of patients undergoing surgery for PHC (1991-2014) was performed. Clinicopathologic characteristics and incidence of PM at the time of index surgery, and one and two years after surgery were compared in patients who did vs. did not undergo TP biopsy. Among 262 patients who underwent surgery, 37 had undergone TP biopsy, and 225 had undergone intraluminal biopsy or had no biopsy. No differences in demographic or clinicopathologic characteristics were noted between groups. The incidence of PM at surgery was not significantly different between TP and non-TP biopsy patients (5.4% vs. 7.6%, p>0.9). Among 243 patients who did not have PM at surgery, the cumulative incidence of PM in the TP and non-TP biopsy groups were not different at one year (11.4% [95%CI 3.5-24.4] vs. 10.8% [95%CI 7.0-15.5]) or two years (20.3% [95%CI 8.7-35.2] vs. 20.1% [95%CI 14.9-25.9]) (p=0.7). Although PM commonly occurs in patients with PHC, TP biopsy was not associated with higher incidence of PM at surgery or at one or two years after surgery.

The Development of Peritoneal Metastasis from Gastric Cancer and Rationale of Treatment According to the Mechanism.

In the present article, we describe the normal structure of the peritoneum and review the mechanisms of peritoneal metastasis (PM) from gastric cancer (GC). The structure of the peritoneum was studied by a double-enzyme staining method using alkaline-phosphatase and 5′-nucreotidase, scanning electron microscopy, and immunohistological methods. The fundamental structure consists of three layers, mesothelial cells and a basement membrane (layer 1), macula cribriformis (MC) (layer 2), and submesothelial connective tissue containing blood vessels and initial lymphatic vessels, attached to holes in the MC (layer 3). Macro molecules and macrophages migrate from mesothelial stomata to the initial lymphatic vessels through holes in the MC. These structures are characteristically found in the diaphragm, omentum, paracolic gutter, pelvic peritoneum, and falciform ligament. The first step of PM is spillage of cancer cells (peritoneal free cancer cells; PFCCs) into the peritoneal cavity from the serosal surface of the primary tumor or cancer cell contamination from lymphatic and blood vessels torn during surgical procedures. After PFCCs adhere to the peritoneal surface, PMs form by three processes, i.e., (1) trans-mesothelial metastasis, (2) trans-lymphatic metastasis, and (3) superficial growing metastasis. Because the intraperitoneal (IP) dose intensity is significantly higher when generated by IP chemotherapy than by systemic chemotherapy, IP chemotherapy has a great role in the treatment of PFCCs, superficial growing metastasis, trans-lymphatic metastasis and in the early stages of trans-mesothelial metastasis. However, an established trans-mesothelial metastasis has its own interstitial tissue and vasculature which generate high interstitial pressure. Accordingly, it is reasonable to treat established trans-mesothelial metastasis by bidirectional chemotherapy from both IP and systemic chemotherapy.

Acacetin inhibits invasion, migration and TGF-\u03b21-induced EMT of gastric cancer cells through the PI3K/Akt/Snail pathway

BackgroundEpithelial-to-mesenchymal transition (EMT) is a pivotal cellular phenomenon involved in tumour metastasis and progression. In gastric cancer (GC), EMT is the main reason for recurrence and metastasis in postoperative patients. Acacetin exhibits various biological activities. However, the inhibitory effect of acacetin on EMT in GC is still unknown. Herein, we explored the possible mechanism of acacetin on EMT in GC in vitro and in vivo.MethodsIn vitro, MKN45 and MGC803 cells were treated with acacetin, after which cell viability was detected by CCK-8 assays, cell migration and invasion were detected by using Transwell and wound healing assays, and protein expression was analysed by western blots and immunofluorescence staining. In vivo, a peritoneal metastasis model of MKN45 GC cells was used to investigate the effects of acacetin.ResultsAcacetin inhibited the proliferation, invasion and migration of MKN45 and MGC803 human GC cells by regulating the expression of EMT-related proteins. In TGF-β1-induced EMT models, acacetin reversed the morphological changes from epithelial to mesenchymal cells, and invasion and migration were limited by regulating EMT. In addition, acacetin suppressed the activation of PI3K/Akt signalling and decreased the phosphorylation levels of TGF-β1-treated GC cells. The in vivo experiments demonstrated that acacetin delayed the development of peritoneal metastasis of GC in nude mice. Liver metastasis was restricted by altering the expression of EMT-related proteins.ConclusionOur study showed that the invasion, metastasis and TGF-β1-induced EMT of GC are inhibited by acacetin, and the mechanism may involve the suppression of the PI3K/Akt/Snail signalling pathway. Therefore, acacetin is a potential therapeutic reagent for the treatment of GC patients with recurrence and metastasis.

Editorial introductions.

Editorial introductions.

Abstract 2380: MiR29b may suppress peritoneal metastasis via the effects on peritoneal mesothelial cells

Abstract Background: Mesothelial cells play tumor-promoting roles in the development of peritoneal metastasis (PM) through mesothelial-mesenchymal transition (MMT). MiR-29 family is well known as tumor suppressor. Methods: MiR-29b mimics and negative control miRNA were transfected by lipofection method. Proliferation and migration of human gastric cancer cell lines, MKN45 and NUGC-4, were examined with MTT assay and transwell assay, respectively. MMT of human peritoneal mesothelial cells (HPMCs) was induced 10 ng/ml of TGF-β. Proliferation and migration of HPMCs were examined with the same methods, and MMT was evaluated with immunofluorescence staining for epithelial, mesothelial and mesenchymal markers. To evaluate the effect of miR-29b in vivo, we developed a highly metastatic subclone YTN16P2 from a murine gastric cancer cell line, YTN16, with in vivo selection, and inoculated the YTN16P2 in peritoneal cavity of syngenic C57BL/6 mice. MiR-29b mimics or negative control miRNA with atelocollagen were intraperitoneally (ip) injected every 3 days from tumor implantation. The mice were sacrificed at 14 days after tumor implantation, and formation of peritoneal metastasis were evaluated. Results: Transfection of miR-29b mimics significantly decreased the proliferation of MKN45 and NUGC-4 by 20-60% (p<0.01) as compared with negative control miRNA. Migration was inhibited more strongly by 90% (p<0.001). After 48 hr-culture with TGF-β, HPMCs apparently changed the morphology from round to spindle shape. The expression level of E-cadherin and Calretinin in HPMCs was decreased while vimentin tended to be upregulated by TGF-β. However, transfection of miR-29b mimics significantly suppressed the morphological changes and reduced the expression levels of vimentin with restored expression of E-cadherin and Calretinin, suggesting the inhibition of MMT in HPMCs. MiR-29b also decreased the proliferation and migration of HPMCs by 20% (p<0.05) and 90% (p<0.001) as compared with negative control miRNA. Finally, mice treated with miR-29b developed less PM nodules than those treaded with negative control miRNA and only atelocollagen. Conclusion: MiR-29b efficiently suppresses MMT. Replacement of the miR-29b in peritoneal cavity might be used for the treatment of PM partially via the effects on mesothelial cell. Citation Format: Yuki Kimura, Hideyuki Ohzawa, Yuki Kaneko, Yurie Futoh, Kohei Tamura, Kazuya Takahashi, Akira Saito, Mineyuki Tojo, Yuko Kumagai, Hideyo Miyato, Naohiro Sata, Joji Kitayama. MiR29b may suppress peritoneal metastasis via the effects on peritoneal mesothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2380.

Isolated effects of regional hyperthermic perfusion on the development of peritoneal metastasis and overall survival in rats with transplanted high-grade ovarian carcinoma

Aim: To construct a perfusion circuit for experimental open hyperthermic intraperitoneal perfusion (HIPEP) and to evaluate the antitumor effects of regional hyperthermia in a model of advanced syngeneic high-grade ovarian carcinoma in vivo. Method: 24 mature female Wistar rats underwent intraperitoneal transplantation of ascitic ovarian tumor 1×107 cells per rat. 48 hours after transplantation the animals were randomized into two groups: I. NIPEP group (12 rats) – normothermic intraperitoneal perfusion (NIPEP) with normal saline at room temperature during 45 minutes; II. HIPEP group (12 rats) – open hyperthermic intraperitoneal perfusion (HIPEP) with normal saline (40.5-41.5 ℃) during 45 minutes. Endpoints included overall survival (OS), the total peritoneal cancer index (PCI), ascites weight and the grade of ascites hemorrhage. Results: In both groups all animals survived the procedure, in the HIPEP group one rat died due to infectious complications on day 32. Compared with NIPEP group HIPEP with normal saline significantly increased the median OS from 19 to 39 days (log-rank test, P=0.0013), reducing the risk of death by 68% (HR=0.32; 95% CI 0.13-0.82). The open HIPEP without a cytostatic was associated with significantly lower total PCI (14 vs 5 points, P=0.0155). In the HIPEP group 3 of 12 animals had intrathoracic tumor spread with malignant pleural effusion without signs of peritoneal carcinomatosis and ascites. Conclusion: The transplanted syngeneic tumor is a valid model that allows to quantitatively assess antitumor activity of intraperitoneal perfusion therapy. Our preclinical data confirmed the role of regional hyperthermia in the treatment of peritoneal carcinomatosis in ovarian tumors.

Association between biopsy method and development of peritoneal metastases in perihilar cholangiocarcinoma

Presenter: Crisanta Ilagan MD | National Institutes of Health Background: In patients with perihilar cholangiocarcinoma (PHC), there is concern that transperitoneal (TP) biopsy may seed tumor in the peritoneal cavity, leading to an increased risk of peritoneal metastases (PM), however, definitive studies are lacking. We aimed to identify any association between biopsy type and development of PM in a cohort of resected patients. Methods: A retrospective review of patients undergoing surgery for PHC (1991- 2014) was performed. The clinicopathologic characteristics and incidence of PM in patients who did and did not undergo TP biopsy were compared. Cumulative incidence of PM was estimated using competing risks methods and compared between subgroups using Gray’s test. Results: Among 262 patients who underwent surgery, 37 had undergone prior TP biopsy, and 225 had undergone intraluminal biopsy or had not undergone biopsy. No differences in demographic or clinicopathologic characteristics were noted between groups. The incidence of PM found at surgery was not significantly different between TP and non-TP patients (5.4% v 7.6%, p>0.9). Among the 243 patients who did not have PM at surgery, the cumulative incidence of PM between TP and non-TP at one year (11.4%, 95%CI 3.5-24.4 v. 10.8% 95%CI 7.0-15.5) and two years (20.3%, 95%CI 8.7-35.2 v. 20.1%, 95%CI 14.9-25.9) was not different (p = 0.7; Figure 1). Cumulative incidence of PM did not differ between patients with positive and negative TP biopsies, positive and negative intraluminal biopsies, and no biopsy performed (p = 0.4). Conclusion: PM occurs at a significant rate in patients with PHC. TP biopsy is not associated with a higher incidence of PM at surgery or cumulative incidence of PM at one or two years after surgery. The risk of peritoneal seeding from TP biopsies may not be as high as case studies suggest, and patients who undergo TP biopsies should be considered candidates for the same treatment modalities as those who do not.

Assessment of circulating VEGF as a predictive biomarker of peritoneal carcinomatosis in gastric cancer

Introduction: Early diagnosis of peritoneal carcinomatosis is difficult in patients with gastric cancer who are at high risk of developing peritoneal metastases. The measurement of serum Vascular Endothelial Growth Factor (VEGF) has proven to be a useful prognostic factor in gastric cancer, but it also could be a predictive factor for peritoneal metastases since the VEGF signalling pathway is directly involved in the development of peritoneal metastases. Methods: This is a retrospective study from 2005 to 2017. We reviewed the peritoneal recurrence pattern of a cohort of 59 gastric cancer patients in whom serum VEGF was measured before surgery and after completion of adjuvant treatment Results: Preoperative serum VEGF (pre-VEGF) level was identified as an independent prognostic factor for developing peritoneal metastases. The optimal cut-off value of pre-VEGF levels was 507 pg/mL, which presented a sensitivity of 66 % and a specificity of 78% to predict the development of peritoneal metastases. Patients with high pre-VEGF levels (>507 pg/mL) were at greater risk of developing peritoneal metastases than patients with low pre-VEGF levels (<507 pg/mL) (p=0.023). Conclusions: VEGF plays a crucial role in the development of peritoneal metastases, and serum VEGF meets the requirements of a potential predictive marker for peritoneal carcinomatosis. Therefore, the measurement of serum VEGF levels could be useful during the follow-up of patients with advanced gastric cancer.

Surgical management and hyperthermic intraperitoneal chemotherapy for locally advanced colorectal cancer.

Locally advanced colorectal cancers (CRC) pose several management challenges, including local recurrence and the development of peritoneal metastases (PM). These recurrences are associated with a poor prognosis and onerous complications. In selected patients with PM, cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CRS-HIPEC) have shown to confer survival benefit. However, its effectiveness depends on the disease extent and this combined treatment is associated with significant morbidity. Additionally, early detection of PM is difficult even with state-of-the-art imaging techniques. Due to the high-risk of developing PM, locally advanced CRC are currently being investigated in several trials, including adjuvant HIPEC in an attempt to reduce the risk of PM. This review article sets out to examine the current data available on this topic, in an attempt to determine the suitability and effectiveness of HIPEC in the management of locally advanced CRC.

Abstract P1-01-07: Clinical and histological characteristics of peritoneal metastases of invasive lobular breast cancer

Abstract Introduction: In previously reported autopsy series, peritoneal metastases have been detected in up to 40% of patients with invasive lobular breast cancer (ILC). Despite modern diagnostic techniques such as high-resolution ultrasound, it remains a challenge to differentiate ovarian cancer from metastatic breast cancer with peritoneal manifestation before or during surgery. The goal of this analysis was to determine typical clinical and immunohistological features of peritoneal metastasis of ILC. Specifically, we asked the question whether there are predictive factors in primary breast cancer associated with subsequent development of peritoneal metastasis. Patients and methods: We identified 58 patients with ovarian metastases in the Charité cancer register (4,792 breast cancer patients from 2003 to 2015). We looked for clinical and pathological differences between breast cancer patients with (N=58) and without (N=4734) peritoneal metastases and between ILC and non-ILC breast cancer subtypes. Imaging and surgical reports of these 58 patients with ILC intraperitoneal metastases were reviewed. Results: The majority (84.7%) of primary breast cancers consisted of subtypes other than ILC and only 15.3% were histologically characterized as ILC. In contrast, 63.6% of patients with peritoneal metastases had histologically proven ILC in the metastatic tissue. Other subtypes where found in the 36.4% of the metastatic tissue (p<0.001). The Odds ratio for peritoneal metastases for ILC was 2.35 (95% CI 1.655-3.332) and for Non-ILC 0.23 (0.185-0.284). There were no significant differences in receptor status between primary and peritoneal metastatic ILC. Comparing ER/PR expressions levels on primary tumor versus metastasis, while statistically not significant (p= 0.805), showed a rise in ER expression in 42.95% in the metastatic tissue while PR expression remained stable with no difference in 53.3% and a rise in the metastatic site in only 26.7% (p= 0.715). Median age of all patients with primary breast cancer was 60 years (10%-90%: 41-75). There was a significant difference in age at diagnosis of metastasis between patients with (50.5 years) and without peritoneal metastases (59 years) (p= 0.002). Median time to development of peritoneal metastases for all patients was 48.5 months (10%-90%: 0-191.7), for ILC 44 months (0-198.2) and for Non-ILC 56.5 months (6.7-206.4) (p= 0.487). Median survival time for patients with ILC and peritoneal metastases was 56 months, for Non-ILC 53 months (p=0.759). 33 of 58 patients had radiologic evidence of disease, 26 with ILC and 7 with Non-ILC. An ovarian mass was detected by imaging in 15/26 patients with ILC and 4/7 patients with non-ILC. Ascites and diffuse peritoneal metastases were seen in 18/26 patients with detectable ILC and in 4/7 patients with Non-ILC. Conclusion: This is the first comprehensive analysis of clinical and pathological characteristics of peritoneal metastases showing ILC is more frequent than other histologic subtypes. Patients with peritoneal metastasis are significantly younger (median 50.5y.) than patients without. The clinical signs are similar to those of ovarian cancer except and therefore the diagnosis of metastatic ILC must be taken into consideration as a differential diagnosis. Citation Format: Karsten MM, Ingold-Heppner B, Oesterreich S, Sander S, Machleidt A, von Waldenfels G, Denkert C, Blohmer JU. Clinical and histological characteristics of peritoneal metastases of invasive lobular breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-01-07.

Abstract 4002: Neutrophil extracellular traps (NETs) in abdominal cavity have supportive roles on postoperative development of peritoneal metastasis

Abstract Background: Peritoneal lavages just after abdominal surgery contain many immune cells which consist mainly of granulocytes. Recent studies have demonstrated that neutrophil extracellular traps(NETs) play important roles not only on capture of pathogenic microbes but also on autoimmune disease, thrombus formation and tumor metastasis. Here, in this study, examined the NET forming capacity of granulocytes in postoperative peritoneal washings and assessed the possible role of the NETs on metastasis formation on peritoneum. Method: In patients who underwent laparotomy due to digestive malignancies, abdominal cavity were washed with 1000 ml of normal saline at the timing of laparotomy (Preoperative) and just before wound closure (Postoperative). Cells recovered from the peritoneal washings were centrifuged on Ficoll-Hypaque solution and cultured at 37c in 5 percentage CO2 condition for 24 hour. Then, extracellular ds-DNA were stained with membrane impermeable dye, SYTOX. Also, the granulocytes were immunostained with mAbs against CD66b, histone, myeloperoxidase (MPO), neutrophil elastase (NE) and Matrix metalloproteinase (MMP)-9, and NETs structures were observed under fluorescein microscopy. In adhesion experiments, human gastric cancer cells were stained by PKH-26 and mixed with the granulocyte culture for 5 min and gently washed. Results: 1. After Ficoll separation, CD66b (+) granulocytes were contained both in polymorophonuclear (PMN) and mononuclear cell (PBMC) layers, which are considered to be normo-density granulocytes (NDG) and low density granulocytes (LDG), respectively. The percentage of LDG in PBMC layer was markedly elevated in postoperative as compared preoperative lavages (Pre: M=0.71%, 0~8.9% vs Post: M=61.3%, 3~86.7% n=39, p<0.001) The LDG in postoperative sample showed multinuclear morphology. 2. The short term culture of the postoperative peritoneal LDG, but not preoperative or peripheral blood granulocytes, many SYTOX (+) fibrous structures were observed, suggesting the vigorous NETs formation. The NETs appeared several min after the incubation, peaked at 2 hour and gradually decreased thereafter. Immunostaining showed that histone and MPO were co-localized on the NETs, while NE or MMP-9 were less clear on NETs. 3. After 5 min co-incubation, human gastric cancer cell, OCUM-1 and MKN45, were selectively attached on the NETs. 4. Similar SYTOX (+) NETs-like structure were clearly detected on the surface of human omental tissue resected by abdominal surgery. Conclusion: After laparotomy, many LDG appear in human abdominal cavity which produce large amounts of NETs on peritoneal surface. The NETs efficiently trap the floating tumor cells, which may have positive roles on peritoneal recurrence in advanced cancer. Citation Format: Rihito Kanamaru, Hidenori Tsukui, Shin Saito, Kentaro Kurashina, Takashi Ui, Shiro Matsumoto, Hironori Yamaguchi, Yasunaru Sakuma, Hisanaga Horie, Yoshinori Hosoya, Naohiro Sata, Joji Kitayama. Neutrophil extracellular traps (NETs) in abdominal cavity have supportive roles on postoperative development of peritoneal metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4002. doi:10.1158/1538-7445.AM2017-4002

Intraperitoneal metastases after transarterial embolization of hepatocellular carcinoma: An observational study.

Transarterial embolization is frequently used to treat local hepatocellular carcinoma (HCC). While various complications are known to occur following transarterial embolization, only one prior case of peritoneal spread of HCC occurring shortly after transarterial chemoembolization has been reported. We present five cases of peritoneal spread of HCC following transarterial embolization (including bland embolization, conventional transarterial chemoembolization (TACE), and doxorubicin-eluting beads TACE) and identify features common among those cases. Search of electronic radiology reports and images identified five patients with imaging before and after treatment of HCC with transarterial embolization and with newly developed peritoneal metastases after treatment. Various patient demographics and tumor characteristics were noted. The mean maximal diameter of the treated HCC tumors was 3.7cm (range 1.4-11.9cm). Three of the patients had ascites and treated tumors in the posterior right hepatic lobe, and all patients had subcapsular tumors treated with transarterial embolization before developing peritoneal metastases. The mean time from treatment with transarterial embolization to the development of peritoneal metastases was four months. Intraperitoneal metastatic disease should be considered a rare but potential complication of transarterial embolization of subcapsular HCC, particularly in patients with ascites and tumors that are in the posterior segments of the right lobe. This potential complication should perhaps be considered when planning transarterial HCC treatment, and radiologists interpreting imaging after transarterial embolization of HCC should assess for peritoneal metastases.

Malignant ascites-derived exosomes promote proliferation and induce carcinoma-associated fibroblasts transition in peritoneal mesothelial cells.

Malignant ascites-derived exosomes have been demonstrated to participate in tumor metastasis. In peritoneal metastasis, normal mesothelial cells (MCs) can be converted into carcinoma-associated fibroblasts (CAFs) by mesothelial-mesenchymal transition (MMT). Herein, we evaluated the effect of malignant ascites-derived exosomes on peritoneal MCs in vitro and in vivo experiments to determine whether exosomes could educate MCs and contribute to peritoneal metastasis.Under the treatment of ascites-derived exosomes, peritoneal MCs showed increased ability to proliferate and migrate. Expression of CAFs specific proteins markers in MCs, including fibroblast activation protein (FAP), alpha-smooth muscle actin (α-SMA), and fibronectin, were increased after treatment of exosomes. In clinical samples test, TGF-β1 was found to be overexpressed in both malignant ascites and malignant ascites-derived exosomes, and the high volume of TGF-β1 may be responsible for peritoneum fibrosis. In addition, exosomes can increase xenograft tumor growth by suppressing the inhibitive ability on tumor cells by MCs. Besides, CAFs specific proteins markers including FAP, α-SMA, and vimentin were increased in clinical peritoneal biopsies. The immunohistochemical staining for mice tumor biopsies also revealed increased expression of fibronectin and FAP, along with decreased expression of E-cadherin and VCAM-1 after exosomes treatment.Thus, malignant ascites-derived exosomes may be of importance in the development of peritoneal metastasis by facilitating MCs to proliferate and convert into CAFs by TGF-β1 induced MMT.

Blocking TGF-β1 by P17 peptides attenuates gastric cancer cell induced peritoneal fibrosis and prevents peritoneal dissemination in vitro and in vivo.

Our previous study demonstrated that the peritoneal stroma environment favors proliferation of tumor cells by serving as a rich source of growth factors and chemokines known to be involved in tumor metastasis. In this study, we investigated the interaction between gastric cancer cells and peritoneal mesothelial cells, and determined the effects of TGF-β1 in this processing. Human peritoneal tissues and peritoneal wash fluid were obtained, which examined by hematoxylin and eosin staining or ELISA for measurements of TGF-β1 levels. The peritoneal mesothelial cells were co-incubated with the supernatants of gastric cancer, the expression of TGF-β1, collagen and fibronectin was observed by ELISA and western blot. We then investigated the effects of serum-free conditioned media from HSC-39 gastric cancer cells on the peritoneum of nude mice, and the effects of peritoneal fibrosis on the development of peritoneal metastasis in vivo. The peritoneum from gastric patients were thickened and contained extensive fibrosis. After co-culture both gastric tumor cells and mesothelial cells, we found that TGF-β1 expression was greatly increased in the co-culture system compared to individual culture condition. Serum-free Conditioned Media from HSC-39 was able to induce extracellular matrix expression in vitro and in vivo, and tumorigenicity in mice with peritoneal fibrosis was greater than in mice with normal peritoneum, while blocking TGF-β1 by peptide P17 can partially inhibit these effects. In conclusion, these results indicated that the interaction of gastric cancer with peritoneal fibrosis and determined that TGF-β1 plays a key role in induction of peritoneal fibrosis, which in turn affected dissemination of gastric cancer.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose.

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.