The Unchartered Fields

Pathology of Peritoneal Metastases The Unchartered Fields

Olivier Glehen Aditi Bhatt Editors

123 Pathology of Peritoneal Metastases Olivier Glehen • Aditi Bhatt Editors

Pathology of Peritoneal Metastases

The Unchartered Fields Editors Olivier Glehen Aditi Bhatt General and Oncologique Surgery Department of Surgical Oncology Centre Hospitalier Lyon-Sud Zydus Hospital Lyon Ahmedabad France India

ISBN 978-981-15-3772-1 ISBN 978-981-15-3773-8 (eBook) https://doi.org/10.1007/978-981-15-3773-8

© Springer Nature Singapore Pte Ltd. 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore Preface

This is a book on pathology of peritoneal metastases that has been edited and largely authored by surgeons which is unusual. Peritoneal surface oncology is a field that has always been at the cross roads—in the early years of evolu- tion of surgical treatment, hyperthermia was being increasingly used to potentiate cancer therapy and thus was combined with the surgical treatment of peritoneal metastases, that is, cytoreductive surgery. The surgery itself had to prove its merit over systemic therapies and was burdened with proving the merit of another treatment that added to the morbidity. Similarly, disease biology was only partially understood and remains a major challenge for future progresses. While the prognostic factors were still being identified, and validated, oncology ushered into the era of genetics and molecular biology. And the gaps in understanding the pathophysiology of peritoneal metastases persisted. Pathological expertise has largely been directed at the diagnosis and classification of uncommon tumors. During cytoreductive surgery that comprises of peritonectomy procedures and visceral resections, a large amount of tissue is submitted for histopathological evaluation. This remains a potential source of prognostic information regarding tumor biology. It provides a good opportunity to also study the patterns and pathways of peritoneal dissemination from various tumors. In this book, we use these pathological findings to better explain the patterns and pathways of peritoneal cancer dissemination and their potential implications on clinical practice. We provide a rationale and recommenda- tions for standardizing CRS procedures and evaluation of surgical specimens. In turn, we raise research question that can be addressed in future studies. Some of the other aspects of pathological evaluation like pathological response to chemotherapy, diagnosis and classification of rare peritoneal tumors have also been covered in different chapters. Keeping in sync with the progress in molecular oncology, we look at the role of molecular oncology in the current and future management of peritoneal metastases. We are grateful to all the contributors for lending their time and expertise to this book. We are also grateful to our pathology colleagues for their invalu- able contribution to this work.

3 December 2019 Olivier Glehen Aditi Bhatt

v Contents

1 Mechanisms of Peritoneal Metastasis Formation �� 1 Yutaka Yonemura, Haruaki Ishibashi, Akiyoshi Mizumoto, Kazuo Nishihara, Yang Liu, Satoshi Wakama, Syouzou Sako, Nobuyuki Takao, Masumi Ichinose, Shun-ichi Motoi, Keizou Taniguchi, Sachio Fushida, Yoshio Endou, and Masahiro Miura 2 Extent of Peritoneal Resection for Peritoneal Metastases: Inferences from Pathophysiology �� 27 Aditi Bhatt and Olivier Glehen 3 Therapeutic Rationale and Data Set for Reporting Cytoreductive Surgery Specimens �� 47 Aditi Bhatt, Nazim Benzerdjeb, Suniti Mishra, and Olivier Glehen 4 Colorectal Peritoneal Metastases: Correlating Histopathological Findings and Disease Biology �� 67 Aditi Bhatt and Olivier Glehen 5 Epithelial Serous Ovarian Cancer: Patterns of Peritoneal Dissemination and their Clinical Implications �� 89 Aditi Bhatt, Loma Parikh, Suniti Mishra, and Olivier Glehen 6 Peritoneal Mesothelioma: Disease Biology and Patterns of Peritoneal Dissemination ��117 Marcello Deraco, Nadia Zaffaroni, Federica Perrone, Antonello Cabras, Shigeki Kusamura, Marcello Guaglio, Matteo Montenovo, and Dario Baratti 7 The Pathological Spectrum of Mucinous Appendiceal Tumours and Pseudomyxoma Peritonei ��131 Aditi Bhatt, Suniti Mishra, Loma Parikh, and Olivier Glehen 8 Genomics in Pseudomyxoma Peritonei ��163 Marco Vaira, Claudio Isella, Michele De Simone, Manuela Robella, Alice Borsano, and Enzo Medico 9 Peritoneal Regression Grading Score (PRGS) for Therapy Response Assessment in Peritoneal Metastasis ��175 Wiebke Solass

vii viii Contents

10 Rare Peritoneal Tumours: Histopathological Diagnosis and Patterns of Peritoneal Dissemination ��181 Suniti Mishra, Snita Sinukumar, Nutan Jumale, Loma Parikh, Aditi Bhatt, and Olivier Glehen 11 Approach to a Patient with Peritoneal Metastases with Unknown Primary Site: Focus on Histopathological Evaluation ��229 Aditi Bhatt, Loma Parikh, Suniti Mishra, and Olivier Glehen 12 Biomarkers in the Management of Peritoneal Metastases ��251 Ninad Katdare, Aditi Bhatt, and Olivier Glehen About the Editors

Olivier Glehen is a world renowned expert in peritoneal surface oncology and a member of the executive committee of the peritoneal surface oncology group international (PSOGI). He is the head of the General and Oncologic Surgery Department at Centre Hospitalier Lyon Sud (Hospices Civils de Lyon) and at the Lyon Sud Charles Mérieux Medical Faculty. His centre is one of centres that have pioneered the surgical treatment of peritoneal metastases in the world. He is director of the Peritoneal Carcinomatosis Research Group from the EMR 3738 (Claude Bernard Lyon 1 University). He has published extensively about peritoneal metastases. He is at the head of RENAPE (French Network on rare peritoneal tumours) and BIG- RENAPE groups (National Clinic-Biological Database on Digestive Peritoneal Carcinomatosis). He is associate editor of European Journal of Surgical Oncology, Journal of Surgical Oncology and Journal of Peritoneum. Professor Glehen is one of the directors of the Inter-University Diploma on Peritoneal Carcinomatosis in France and his centre is a reference centre for the European Society of Peritoneal Surface Oncology (ESPSO) certified fel- lowship in peritoneal surface oncology. His centre performs more than 200 cytoreductive surgery and HIPEC (Hyperthermic Intraperitoneal Chemotherapy) procedures a year and is also one of the leading centers in the world that is developing PIPAC (Pressurized Intraperitoneal Aerosol Chemotherapy).

Aditi Bhatt is an Indian surgical oncologist specializing in the management of peritoneal surface malignancies with an experience of 10 years in the same. She is one of the founding members of the Society of peritoneal surface oncology, India, the Indian HIPEC registry and serves as the honorary secre- tary of the Asian Peritoneal Surface Malignancy Group. She has published several scientific papers on the subject, edited two special issues of the Indian Journal of Surgical Oncology on the same and edited a book on peritoneal surface malignancies.

ix Mechanisms of Peritoneal Metastasis Formation 1

Yutaka Yonemura, Haruaki Ishibashi, Akiyoshi Mizumoto, Kazuo Nishihara, Yang Liu, Satoshi Wakama, Syouzou Sako, Nobuyuki Takao, Masumi Ichinose, Shun-ichi Motoi, Keizou Taniguchi, Sachio Fushida, Yoshio Endou, and Masahiro Miura

1.1 Introduction proximal distribution” by Sugarbaker [3] (Fig. 1.1). Through the process, cancer cells with It has long been considered that the establishment high malignant potential can metastasize on the of peritoneal metastasis (PM) is a multi-step pro- peritoneum by concerted expression of metasta- cess, consisting of (1) detachment of cancer cells sis-related genes [1–3]. Recently, new concepts of from the primary tumor, (2) adhesion of perito- the formation of PM were proposed: i.e., (1) neal free cancer cells (PFCCs) on the distant peri- Trans-lymphatic metastasis and (2) superficial toneal surface, (3) invasion into the submesothelial growing metastasis (Table 1.1) [3]. tissue, and (4) proliferation accompanying with In this chapter, mechanisms of the formation the angiogenesis and the induction of stromal tis- of PM will be described in terms of the morpho- sue [1]. The process is called “trans-mesothelial logical, histological, and molecular biological metastasis” by Yonemura et al. [2] or “randomly aspects.

A. Mizumoto · N. Takao · M. Ichinose · S.-i. Motoi Department of Regional Cancer Therapy, Peritoneal Dissemination Center, Kusatsu General Hospital, Y. Yonemura (*) Kusatsu, Shiga, Japan Asian School of Peritoneal Surface Malignancy Treatment, Kyoto, Japan K. Taniguchi Department of Surgery, Mizonokuchi Hospital, Department of Regional Cancer Therapy, Peritoneal Teikyo University, School of Medicine, Dissemination Center, Kishiwada Tokushukai Kawasaki, Kanagawa, Japan Hospital, Kishiwada, Osaka, Japan S. Fushida Department of Regional Cancer Therapy, Peritoneal Department of Surgery, Kanazawa University, Dissemination Center, Kusatsu General Hospital, Kanazawa, Japan Kusatsu, Shiga, Japan e-mail: [email protected] Y. Endou Central Research Resource Center, Cancer Research H. Ishibashi · K. Nishihara · Y. Liu · S. Wakama Institute, Kanazawa University, Kanazawa, Japan S. Sako Department of Regional Cancer Therapy, Peritoneal M. Miura Dissemination Center, Kishiwada Tokushukai Department of Anatomy, Oita Medical University, Hospital, Kishiwada, Osaka, Japan Oita, Japan

Rolling, cross Invasion Invasion into Adhesion to talk into angiogenesis Establishment of Cancer cells the gap of basement with mesothelial submesothelial peritoneal MC cells membrane cell (MC) tissue metastasis

HIF, VEGF EGF/EGFR, Gene CD-44, CA19-9 Integrin UPA/UPAR AMF/AMFR, TGF- , PIGF MET products CEA, α2,3,5 MMPs, MT- β MET REG IV/Reg from P-cadherin β1,2 MMPs Rho, S1004A Angiogenesis receptor cancer cells Integrin, Motility factors Adhesion mol. Motility factors Heterophilic Matrix digesting factors Growth factors and adhesion enzymes their receptors molecules Peritoneal PFCC Macrophage cavity

Mesothelial cell

Blood peritoneal barrier 90µm

Blood Blood vessel Fibroblast vessel CD34

ICAM HGF, EGF Macrophage Gene HGF, EGF VCAM TGF-β MMP-8, -9 VEGFR-1,2, Tie-2, products TGF-β from PECAM-1 Fibroblast TGF-β E-selectin Contraction of MMP-1,-2,-3 CD144, CD31 stromal Fibroblasts cells Hyaluronic acid MC PIGF, plasmin MC injuring Ephrin, VE-cadherin Mesothelial cells factors (MC) Proliferation of Basement membrane vascular endothelial cells Macula cribriformis

Fig. 1.1 Trans-mesothelial metastasis as named by of basement membrane and macula cribriformis (Process Yonemura et al. or randomly proximal distribution by 2). PFCCs then migrate into the inter mesothelial space, Sugarbaker. Peritoneal free cancer cells (PFCCs) exfoli- and invade into the submesothelial tissue by degradation ated from the serosal surface of primary tumors migrate of extracellular matrix by matrix-digesting enzymes and into the peritoneal cavity, and adhere on the peritoneal by locomotive activity using motility factors (Process 3). surface (Process 1). During the rolling of PFCCs on the Finally, cancer cells proliferate near the submesothelial peritoneal surface, peritoneal mesothelial cells shrink by blood vessels with introduction of tumor stromal tissues cytokines produced by PFCCs, resulting in the exposure and neogenesis of tumor blood vessels (Process 4)

Table 1.1 Three patterns of peritoneal metastasis according to the biological malignant potentials and the morphological feature of peritoneal free cancer cells Biologic malignant Morphological features of Pattern of metastasis behavior Cancer PFCCs Trans-mesothelial metastasis High Gastric, colorectal, pancreatic, Single or small clusters biliary ovarian cancer Trans-lymphatic metastasis High, moderate Gastric, colorectal, ovarian Single or small clusters Omental milky spots (OMS) cancer Initial lymphatics outside OMS Superficial growing metastasis Moderate, low AMNa, GCSb, mesothelioma, Large with or without MCMc, hepatoma mucinous material aAMN low-grade appendiceal mucinous neoplasm bGCS granulosa cell tumor cMCM multicystic mesothelioma 1 Mechanisms of Peritoneal Metastasis Formation 3

1.2 Mechanisms of Trans- ence junction on the basolateral membrane side mesothelial Metastasis (Fig. 1.2). Tight junction components are transmembrane proteins, claudin and occludin and the 1.2.1 Mechanisms of Cancer Cell cytoplasmic scaffolding protein, ZO-1, -2, and Spillage into the Peritoneal -3, which bind the actin bundles of the cytoplas- Cavity mic protein. Tight junction is mainly composed of claudin, which is observed as continuous bead- The first step of PM is detachment of cancer cells like particles expressed on the lateral membrane from the serosal surface of the primary tumor in of cells [4]. The partition of the basilar membrane highly malignant tumors and the rupture of by the tight junction functions as a barrier to con- appendix vermiformis or ovary by the increased trol the movement of materials and as a selective intrinsic pressure due to the proliferation on permeation channel [4]. However, dysfunction of mucinous neoplasm. Additionally, during sur- the tight junction causes the loosening of cell-cell gery, blood or lymphatic fluid contaminated with adhesion, resulting in edema. Disappearance of cancer cells may spill into the peritoneal cavity tight junction causes cells to disperse by the loss from damaged blood and lymphatic vessels. The of polarity. Vascular endothelial growth factor exfoliated cancer cells in the peritoneal cavity are (VEGF) and some cytokines reduce the function called peritoneal free cancer cells (PFCCs). of tight junction, and cause edema [5]. PFCCs have high proliferative activities and can Serial analyses of gene expression (SAGE) grow in the distant peritoneum. clarified the reduced expression of claudin in In the process of detachment of cancer cells poorly differentiated adenocarcinoma of the from the primary tumor, homophilic cell-cell stomach [6], in which reduced expression of adhesion molecules play important roles. claudin 4, 7 is found and the patients with gastric Epithelial cells tightly interconnect with a tight cancer showing low expression of claudin have a junction on the apical membrane and an adher- poor prognosis. In addition, ZO expression is

Fig. 1.2 Molecules associated with homophilic cell-cell adhesion of epithelial cells. Epithelial cells JAM interconnect with tight junction, located at ZO-1, -2, -3 apical membrane of Claudin cells, and with Tight junction ZO-1, -2, -3 adherence junction on the basolateral ZO-1, -2, -3 membrane. Cancer cells showing the functional Occludin abnormalities of tight Nectin junction and adherence Actin bundle junction tend to disperse and detach from the Adherence Afadin serosal surface, spill junction from the primary tumor ADIP Ponsin and migrate into the α-Actinin Vinculin peritoneal cavity β-catenin α-catenin

P120 Cadherin ctn

Basement membrane 4 Y. Yonemura et al. also reduced in poorly differentiated adenocarci- adhesion molecules (ICAM-1, CD54) [16] and noma which tends to establish PM [6, 7]. vascular cell adhesion molecules (VCAM-1, In the components of the adherence junction, CD106) [17], and interact with integrin αLβ2 E-cadherin expression is important. E-cadherin is (LFA-1α, CD11a) expressed on PFCCs and a transmembrane glycoprotein, and cells tightly inflammatory cells (heterotypic cell-cell connect with the extracellular domain of the mol- adhesion). ecule on the basolateral membrane (homophilic Platelet-endothelial cell adhesion molecules adhesion). The intracellular domain of E-cadherin (PECAM-1, CD31) play a role in the transmigra- connects with α-, β-, γ-catenin and the complex tion of white blood cells and PFCCs. After the controls the function of E-cadherin [8–10]. In process of slowing down the passage of leuko- gastric cancer, poorly differentiated adenocarci- cytes and PFCCs over activated vascular endo- noma especially macroscopic type-4 has a high thelium and peritoneal mesothelial cells potential of PM and the downregulation of (“rolling”), integrins are crucial for stopping the E-cadherin expression is an important feature cells at the extravasation site and migration into [11, 12]. The causes of reduced expression of submesothelial tissue [18]. E-cadherin are loss of the E-cadherin gene, point Stopping is the result of the interaction of inte- mutation, and the methylation of the promoter grins on the leukocytes (β2 integrin, α4β1 (VLA- region [12]. In addition, the function of 4), or α4β7) and immunoglobulin-like adhesion E-cadherin is reduced via the inhibition of the molecules (ICAM-1 and VCAM-1) on endothe- function of catenin molecules by the loss of the lial and mesothelial cells [19]. Next, for migra- α-catenin gene, and phosphorylation of the tyro- tion based on the interaction between β2 integrins sine residue of β-catenin [13]. In colorectal can- on the leukocytes and ICAM-1, VCAM-1, and cer, downregulation of E-cadherin level is PECAM-1 on the endothelial cells, mesothelial associated with poorly differentiated type, higher cells adhere with inflammatory cells or PFCCs potential of metastasis, and progression [14]. The via homophilic adhesion of PECAM-1. The lower expression of E-cadherin in poorly differ- expression of VCAM-1, and E-selectin, is upreg- entiated colorectal cancer may explain the ulated by IL-1β, TNF-1α, and IFN-γ, so the aggressive nature, and poorly differentiated ade- mesothelial cell adhesion can be facilitated by nocarcinoma is known as a subtype that fre- local inflammation [16, 20]. quently metastasizes on peritoneum [14, 15]. As shown in Fig. 1.3, several adhesion mole- Accordingly, cancer cells with the functional cules are associated with the adhesion between abnormalities of tight junction and adherence mesothelial cells and PFCCs. The only classical junction tend to disperse and detach from the cadherin expressed by mesothelial cells and serosal surface, spill from the primary tumor and PFCCs is P-cadherin, and this molecule could migrate into the peritoneal cavity. serve homophilic heterotypic adhesion (heterophilic adhesion by the same adhesion molecule) between PFCCs and mesothelial cells (Fig. 1.3) 1.2.2 Adhesion of PFCCs [21]. P-cadherin promotes intraperitoneal dis- and Mesothelial Cells (Fig. 1.1, semination of ovarian cancer cells by facilitating Process 1) tumor cell aggregation and tumor peritoneum interaction in addition to promoting tumor cell PFCCs migrate on the distant peritoneal surface migration [22]. and adhere to mesothelial cells during rolling on Hyaluronic acid (hyaluronate) is a mucopro- the mesothelial cell surface. PFCCs express sev- tein with a molecular weight of 200,000–400,000, eral kinds of integrin molecules and adhere with consisting of alternate binding of their ligands expressed on mesothelial cells N-acetylglucosamine and glucuronic acid. This (Fig. 1.1, Process 1, 2). Peritoneal mesothelial molecule is one of the components of extracellu- cells express immunoglobulin-like intercellular lar matrices and acts as a cushion to support the 1 Mechanisms of Peritoneal Metastasis Formation 5

actin Peritoneal free cancer cell

TNFα

P-cadherin α4β1 α4β7 αLβ2 α4β7 CD44 L1CAM IL-1β Sialyl Lex CA125

macrophage VCAM-1 CD56 ICAM-1 P-cadherin E-Selectin Inflammatory cell CD106 hyaluronate TNFα fibroblast mesothelin IL-1β

Mesothelial cell Neuropirin-1

Fig. 1.3 Heterotypic adhesion molecules associated with adhesion between cancer cells and mesothelial cells cells by binding a considerable amount of water. The sialyl Lewis-a (sLea) antigen is a carbohy- Pericellular hyaluronate produced by mesothelial drate structure present on cancer cells and has a cells has a function as a lubricant (Fig. 1.3). structure of sialic form of Lea antigen with sialyl CD44 is a transmembrane glycoprotein and acts acid. The monoclonal antibody CA19-9 is a use- as a receptor for hyaluronate. This molecule has ful and popular tool to assess circulating sialyl four functioning domains, and hyaluronate binds Lewis-a epitopes in the blood of cancer patients. with the extracellular domain of CD44. PFCCs sLea expressed on leukocytes binds to P-cadherin expressing CD44 bind to the pericellular hyal- on the activated vascular endothelial cells, and uronate of the mesothelial cells. The CD44 gene the weak affinity interaction of sLea and has 20 exons and many translational products P-cadherin is considered as the initial force (v1–v10) are produced from alternative RNA implicated in the “rolling” of extravasating leu- splicing and post-translational modifications. kocytes. E-selectin also can bind to sLea, and sLea Certain CD44 isoforms that regulate activation on cancer cells produces a similar interaction and migration of lymphocytes and macrophages with E-selectin on the endothelial cells [27]. may also enhance local growth and metastatic As shown in Fig. 1.3, E-selectin is also found spread of tumor cells [23]. CD44v, v7 splice vari- on peritoneal mesothelial cells. The interaction ants are expressed by some gastro-intestinal can- of E-selectin and P-cadherin on mesothelial cers, and considered as markers for their cells and sLea on PFCCs has a role on the het- metastatic capability [24]. CD44v6 expressed on erotypic adhesion in early pathophysiological PFCCs accounts for the binding with mesothelial event in PM. cells [24]. The TGF-β produced from the fibro- CA125 is a cell surface mucin-like glycopro- blasts in the stroma of cancer tissue upregulates tein expressed in mesothelial cells, and is upregu- the CD44 expression from cancer cells [25]. lated in malignant ovarian tumors [28, 29]. It is Expression of CD44 by peritoneal mesothelial considered as a relatively specific circulating cells also seems to contribute to heterotypic cell tumor marker in ovarian cancer patients. adhesion by pancreatic cancer cells, and is upreg- Mesothelin is expressed by the normal mesothe- ulated by TNF-α/IL-1β from the peritoneal mac- lial cells, and soluble mesothelin is used to detect rophages [26]. the overexpressed protein as a circulating tumor 6 Y. Yonemura et al. marker for mesothelioma. Mesothelin binds with shock proteins, granulocyte-colony stimulating CA125 expressed on PFCCs and has a role as an factor (G-CSF), IL-15, IL-1β, TNF-α, and epi- adhesion molecule between ovarian cancer cells dermal growth factor (EGF) to have cross-talk, and mesothelial cells [30]. and these substances immediately react with the L1CAM (CD171), an adhesion molecule changes in the peritoneal environment [34, 35]. expresses on pancreas cancer and colorectal can- When PFCCs contact with mesothelial cells, cer cells and binds with neuropilin-1 (Fig. 1.3). It many changes are found in the mesothelial cells is upregulated by TGF-β1 [31]. Soluble L1CAM (Figs. 1.4 and 1.5). (sL1) binds to VEGF-A (165), and activates Akedo et al. reported three growth patterns of VEGFR-2, resulting in angiogenesis [32]. cancer cells are found when rat hepatoma cells were co-cultured with a rat mesothelial monolayer [36]. Tumor cells either formed “pile-up” 1.2.3 Morphological Changes nests upon the mesothelial monolayer, exhibited of Mesothelial Cells (Fig. 1.1, invasive growth between adjacent mesothelial Process 1), Submesothelial cells (flattened tumor cell island), or failed to Invasion of PFCCs, Attachment attach and grew in suspension. of PFCCs to the Basement When ascitic fluid was added into the medium Membrane (Fig. 1.1, Process 2) of the mesothelial monolayer, the mesothelial cells took up a characteristic “round” morphol- Mesothelial cells are flat and squamous-like cells ogy with separation of cell–cell contacts after and connect to each other with a tight junction. The diameter of mesothelial cell is approximately 25 μm (Fig. 1.4). Mesothelial cells provide a protective barrier against invading pathogens and PFCCs. The surface of activated mesothelial cells has a well-developed microvilli varying in length, shape, and density (Fig. 1.5). Cilia are also present on some resting mesothelial cells, but are more abundant on activated cells. They may be part of a sophisticated surveillance system that may respond to elicit discrete cellular responses [33]. ICAM-1 and VCAM-1 are expressed on the microvilli of mesothelial cells, and the materials correlate with the cross-talk of cells expressing Fig. 1.5 Activated human mesothelial cells. Microvilli integrins. Mesothelial cells produce IL-6, IL-1 expressed on cell surface of human mesothelial cells from (expressed by bacterial lipopolysaccharide), heat resected specimens from pseudomyxoma peritonei

abc

Fig. 1.4 Morphological changes of mesothelial cells. (a) Normal, (b) shrinkage of cytoplasm, (c) separation and exposure of submesothelial basement membrane (human mesothelial cells) 1 Mechanisms of Peritoneal Metastasis Formation 7

20 h [37]. These results denote that the malignant 8β subunits, 24 kinds of integrins exist [39]. Many ascitic fluid contains factors that induce the kinds of integrins are expressed from PFCCs, and changes of mesothelial cell morphology (meso- the overexpression of integrins correlates with thelial cell injury factors). These factors are metastatic potential [40–42]. Integrin α2 and α3 produced from cancer cells, peritoneal macro- expressions were significantly elevated in the phages, and mesothelial cells [38]. peritoneal dissemination of gastric cancer [40, 41]. These α-integrins dimerize with β-subunits to form adhesion molecules for basement membrane 1.2.4 Adhesion of PFCCS proteins, including fibronectin, laminin, and col- to the Submesothelial lagen IV. Treatment with anti-β1 integrin antibody Basement Membrane (Fig. 1.1, significantly inhibited the adherence of highly Process 2 and Fig. 1.6) metastatic cell line on the peritoneum in an ex- vivo peritoneal model, suggesting a role for After mesothelial cell contraction by cytokines, β1-mediated integrin adhesion to the submesothe- the submesothelial basement membrane is lial basement membrane [41]. In ovarian cancers, exposed (Fig. 1.4). The basement membrane con- integrin α5β1 and α6β1 correlate with PM. sists of laminin, type IV collagen, heparin sulfate proteoglycan, entactin, and perlecan. Mesothelial cells and fibroblasts produce these elements. 1.2.5 Invasion into Current evidence suggests that adherence to the the Submesothelial Tissue basement membrane of PFCCs is mediated via (Fig. 1.1, Process 3) an integrin-ligand interaction. The integrin molecule is a heterodimer consist- Factors associated with invasion into the subme- ing of an α and a β subunit and is expressed on the sothelial tissue are the autocrine motility factor cell membrane. Integrins are the important mole- (AMF)/AMF receptor, Rho/ROCK, S100A-4, cules for cell-cell and cell-ECM adhesion. and hepatocyte growth factor (HGF)/MET According to the combination of 17α subunits and (receptor for HGF) [43–48].

Fig. 1.6 Highly metastatic cell line (MKN-45) from gastric cancer express filopodia, and attach to the basement membrane of human greater omentum 8 Y. Yonemura et al.

AMF is a 55 kDa protein, which stimulates tyrosine kinase type receptor) are important mol- chemotaxis and chemokinetics [49]. AMFR is a ecules for cell motility and proliferation. When member of the tyrosine kinases, which are located HGF binds with MET, MET is activated by the on the cell membrane. Binding of AMF with autophosphorylation of tyrosine residue on the AMFR stimulates changes in the cytoskeleton intracellular domain and induces cell motility by and formation of invadopodia, resulting in the activation of F-actin and microtubules. In the induction of amoebic movement [43]. Type 4 peritoneal cavity, HGF is produced from acti- gastric cancer is more significantly associated vated mesothelial cells, and fibroblasts, and with PM than the other macroscopic types. In induces mesothelial cell contraction and invasion type 4 gastric cancer, expression of the AMFR of PFCCs through the intercellular space of protein was significantly higher than that in type mesothelial cells [48]. 3 tumors [50]. Accordingly, poorly differentiated IL-1β, and TNF-α from peritoneal macro- adenocarcinoma of the stomach has high motility phages, fibroblasts, and inflammatory cells by the activation of the AMF/AMFR cascade induce HGF production from mesothelial cells combined with downregulation of E-cadherin [51]. The HGF/MET paracrine cascade corre- and claudin [7, 12, 50]. lates with not only cancer cell motility but also Rho is a G protein, which induces ruffling of proliferation and angiogenesis. Recently, molec- the cell membrane in cooperation with effectors ular targeting therapy to control the cascade has of its downstream, like mDia, Crk, Rac and been developed [52]. ROCK, and FAK/paxillin [49]. Rho upregulates actin filaments by activation of mDia, and ROCK increases the contractile strength of myosin, 1.2.6 Destruction of Submesothelial which bridges actin filaments. Rho and Rac Basement Membrane expressions were upregulated in poorly differen- and Extracellular Matrix (ECM) tiated adenocarcinoma and advanced cancers in and Invasion into the late stage [43]. Submesothelial Tissue (Fig. 1.1, S100A4, a member of the S100 protein fam- Process 3) ily, is known as a calcium-binding protein, and increases cell motility by activating myosin [44]. The tissue between mesothelial cells and subme- S100A4 activates myosin in lamellipodia sothelial arterial blood capillaries is named the expressed on the invasion front of cancer cells peritoneal-blood barrier, and the average width is (Fig. 1.6) [45]. The actin filament that bridges 90 μm (Figs. 1.1, 1.17 and 1.29) [53]. This barrier myosin, combined with the vinculin connected prohibits the diffusion of drugs administered by with talin and the intracellular domain of integrin systemic chemotherapy. The diffusion length of [45, 46]. In gastric cancer, S100A4 upregulation oxygen from arterial blood capillaries is 100 μm, is significantly associated with poorly differenti- and PFCCs attached to the submesothelial base- ated adenocarcinoma, lymph node metastasis, ment membrane can survive by the oxygen nutri- peritoneal dissemination, and a poor prognosis tional supplement from blood vessels [54]. [46]. In addition, downregulation of E-cadherin PFCCs with high invasive capacity destroy the and upregulation of S100A4 were found in type 4 ECM in peritoneal-blood barrier, invade near the gastric cancer [46]. Moriyama et al. reported that arterial blood capillaries, and proliferate with S100A4 gene was transfected into a non-invasive angiogenesis (Fig. 1.1, Process 4). oral cancer cell line of OSC-19, and that the new The subperitoneal basement membrane cell line overexpressed S100 A4, showed signifi- between mesothelial cells and submesothelial cant invasive activity, and downregulated stromal tissue is a thin membrane of 50–100 nm E-cadherin and β-catenin [47]. in width, and is composed of collagen type IV, The scatter factor (SF) called hepatocyte laminin, entactin, heparin sulfate proteoglycan, growth factor (HGF) and its receptor of MET (a and perlecan [55, 56]. Molecules associated 1 Mechanisms of Peritoneal Metastasis Formation 9 with the destruction of basement membrane are MMP genes are upregulated by IL-1, TNF-α, matrix metalloproteinases [MMPs: MMP-2, EGF, PDGF, and FGF. MMP-1, -2, -7, -13, and MMP-7, MMP-14 (MT1-MMP)] and plasmin. -14 (MT1-MMP) play roles in the stromal inva- These molecules are produced from cancer sion of gastric cancer. cells, mesothelial cells, fibroblasts, inflamma- MMP-1 specifically cuts the helix structure of tory cells, and macrophages. Subperitoneal tis- collagen types I, II, and III. In gastric cancer tissues are composed of dense network of ECM, sue, MMP-1 is secreted from the stromal fibro- which prohibits the movement of materials with blasts. TGF-β produced from poorly differentiated molecular weight higher than 100,000. Cancer adenocarcinomas of the stomach stimulates the cells produce several kinds of matrix-digesting proliferation of fibroblast in the invasive front enzymes to destroy and invade into subperito- [61]. Cancer cells invade the stroma utilizing neal stromal tissue. An immunohistochemical MMP-1, and MMP-2 produced from the fibro- study of gastric cancers revealed that urokinase- blasts. TGF-β inhibits the proliferation of the epi- type plasminogen activator (UPA) is detected in thelial cells. In contrast, TGF-β II receptor the cytoplasm in 66% of gastric cancers [57]. expression is downregulated in type 4 gastric UPA from gastric cancer and fibroblasts binds cancer, which evades the inhibition of prolifera- with its receptor (UPAR) on the cell membrane tion by TGF-β from fibroblasts [61]. and is activated with plasmin and kallikrein. MMP-2 (gelatinase A) degrades gelatin, col- Activated UPA on the cell membrane activates lagen types IV, V, VII, X, and XI, fibronectin, plasminogen to plasmin [58]. Plasmin then elastin, and proteoglycan, which are components degrades the ECM and further activates plas- of the ECM [52]. TIMP-2 combines with acti- minogen and latent MMPs. UPA is specifically vated MMPs and proMMP-2, and controls the inactivated by plasminogen activator inhibitor-2 activity and degradation of MMP-2. ProMMP-2 (PAI-2), and PAI-2 can inhibit the formation of (72 kDa) when activated by MT1-MMP becomes experimental peritoneal carcinomatosis [59, active MMP-2 (62 kDa), which activates MMP-9 60]. UPAR expression in type 4 gastric cancers and MMP-13, resulting in the degradation of is significantly higher than that in other macro- many kinds of ECM components. scopic types [57]. Fibroblasts accumulate in the MT-MMP is detected on the cell membrane stroma of the invasive front of type 4 gastric (Fig. 1.7) and plays roles in cell migration, cancer. UPA secreted from fibroblasts is combined with UPAR on the cancer cells via the paracrine loop, leading to activation of plasmin in the cancer cells which help them to invade the stomach wall [60]. MMP family includes collagenases (MMP-1, MMP-8, and -13), gelatinases (MMP-2 and MMP-9), stromelysin-1, -2 (MMP-3, and MMP- 10), transmembrane MMPs (MT-MMP families), and others: matrilysin, MMP-7; stromelysin-3, MMP-11; metalloesterase, MMP-12; and enam- elysin, MMP-20. Activities of MMPs are con- trolled by the activation of proMMPs and inhibition by TIMPs (tissue inhibitor metalloproteinases), and MMPs are mutually activated by plasmin and the other MMPs. Four types of TIMPs have been reported and control the activ- Fig. 1.7 MT1-MMP expression on the invadopodia. Immunofluorescent staining with anti-MT1-MMP mAb ity of MMPs, resulting in the degradation of col- for TMK-1 cells (gastric cancer cell line), transfected with lagen and the induction of fibrosis. MT1-MMP gene 10 Y. Yonemura et al. differentiation, and morphological change by those without MMP-13 expression. Patients with the degradation of the pericellular ECM. The tumor expressing both MMP-13 and MT1-MMP MT-MMP family has 6 kinds of molecules showed the worst prognosis [60]. (MT1-6-MMP). MT1-MMP forms a homo-oligomer on the pseudopodia of cancer cells, and induces an effi- 1.2.7 Proliferation cient invasion by the degradation of their pericel- in the Subperitoneal Tissue lular ECM [53]. MT1-MMP itself degrades (Fig. 1.1, Process 4: collagen types I, II, and III, fibronectin, laminin, Angiogenesis vitronectin, and aggrecan, and plays a role in the and Proliferation) activation of proMMP-2 [54]. TIMP-2 combines with a catalytic domain of MT1-MMP. A com- Tyrosine kinases play a major role in the prolif- plex of TIMP-2-MT1-MMP binds with eration of cancer cells. The interaction of the proMMP-2 and forms a tertiary complex. growth factors with the receptors activates signal- ProMMP-2 becomes an intermediate active ing pathways and induces mitogenesis. Among MMP-2 by the activation of neighboring MT1- these receptors, K-sam, EGFR, MET, vascular MMP [55]. In poorly differentiated gastric endothelial growth factor receptor (VEGFR), and cancers, the MMP-2 secreted from fibroblasts is ERBB are frequently involved in PM of various activated by MT1-MMP. A paracrine loop of cancers. In gastric cancers, expressions of K-sam, MMP-2 from fibroblasts and MT1-MMP on gas- EGFR, MET, and VEGFR are associated with tric cancer induces invasion and metastasis of proliferation and angiogenesis. gastric cancer [56]. The K-sam gene encodes the receptors against MMP-7 (matrilysin) itself degrades collagen fibroblast growth factor (FGF) and keratinocyte types I, II, III, and IV, aggrecan, laminin, and growth factor (KGF). When the K-sam gene fibronectin, and can activate proMMP-1, -3, -8, product is activated, the ras-raf-MAP kinase cas- and -9 secreted from cancer cells and fibroblasts. cade is activated and cell proliferation is induced As a result, almost all ECM components can be [63]. In type 4 gastric cancer, K-sam gene ampli- degraded by MMP-7. A study of serial analyses fication is a characteristic feature. In the poorly of gene expression of gastric cancer revealed the differentiated types of gastric cancer, expression overexpression of MMP-7 [57]. A highly meta- of bFGF for the ligand of K-sam is significantly static cell line (MKN-45-P) on the peritoneal sur- upregulated, and cancer cell proliferation is stim- face overexpressed MMP-7 [58]. Intraperitoneal ulated by the autocrine or paracrine loop [64]. In administration of an antisense oligonucleotide an immunohistochemical study of keratinocyte against MMP-7 mRNA improved the survival of growth factor (KGF) and K-sam expression, the the mice bearing MKN-45-P [59]. The incidence incidence of K-sam expression was significantly of MMP-7 protein expression in the type 4 gas- higher in type 4 gastric cancer than in the other tric cancer is significantly higher than that of the types. In addition, patients with tumor coexpress- other macroscopic types [43]. ing K-sam and KGF had significantly poorer MMP-13 is produced from cancer cells and prognosis [65]. Accordingly, the paracrine loop chondrocytes and degrades collagen types I, II, of K-sam/KGF/bFGF has an important role in the and III. MMP-13 mRNA was expressed in 8 of 9 progression of gastric cancer, especially in poorly gastric cancer cell lines, and in these cell lines differentiated type and type 4 gastric cancer. MMP-13 mRNA was coexpressed with MMP-2 The epidermal growth factor receptor (EGFR) and MT-1 MMP, which activate proMMP-13 [60]. and its family of Her-2/ERBB-2, Her-3, and MMP-13 mRNA expression was found in 61% of Her-4 are upregulated in 70% of all cancers [66, gastric cancer patients in stage IV disease [62], 67]. Signals of EGFR are transduced through the and the prognosis in patients with MMP-13 over- ras-raf-MAP kinase route, PI3K-Ak route, and expressing tumor was significantly poorer than in Jak-STAT route, and they induce proliferation, 1 Mechanisms of Peritoneal Metastasis Formation 11 growth, and apoptosis. Type 4 gastric cancer co- angiogenesis [74], and the expression of vascular expresses EGF and EGFR [68]. endothelial growth factor (VEGF) is stimulated MET is associated with not only cell scatter- [75, 76]. VEGF binds with VEGF receptor-1 and ing, but also proliferation [69]. In gastric cancer, stimulates endothelial cell proliferation. Almost alteration of the domain induces a constant acti- all cancer cells produce VEGF, which has a major vation of the downstream components [70]. In role in the establishment of PM [77]. VEGF-C, addition, c-met gene amplification is detected in which is a specific molecule for lymphangiogen- gastric cancer, and upregulates the signal trans- esis, activates VEGFR-3 (flt-4) [64]. duction downstream of MET activated in a ligand-dependent or non-ligand-dependent man- ner [71]. Many molecular targeting strategies to 1.3 Trans-lymphatic Metastasis decease MET function by controlling Try 1003 have been studied [72, 73]. In 2012, Yonemura reported a new concept of PM When the diameter of a cancer nest is greater formation, called trans-lymphatic metastasis [78] than 100 μm, oxygen and nutritional supplemen- (Fig. 1.8). Trans-lymphatic metastasis is the met- tation from preexisting blood vessels are not astatic pathway by which the PFCCs migrate into sufficient for survival of cancer cells. Accordingly, the submesothelial initial lymphatic vessels cancer cells that are located more than 100 μm through mesothelial stomata (Fig. 1.9), and holes apart from blood vessels will die off. of macula cribriformis (Fig. 1.10) [78]. In such a situation, angiogenesis is induced by Subperitoneal lymphatic vessels associated with angiogenic factors secreted from cancer cells, trans-lymphatic metastasis are found in omental and cancer tissue with newly formed vessels can milky spots (OMS) and initial lymphatic vessels be established. Proliferating cancer cells upregu- in parietal peritoneum, and small bowel late hypoxia inducible factor (HIF)-1α to induce mesentery.

Mesothelial gap, stomata Macura cribriformis Basement stomata membrane

Cribriform plate Initial Flat type lymphatic Protruded vessel type

Submesothelial lymphatic vessel

Blood vessel

Fig. 1.8 Initial lymphatic vessels, which directly connect pouch, paracolic gutter, and small bowel mesentery. The with peritoneal cavity via mesothelial stomata and hole of latter is detected on the pelvic peritoneum. PFCCs macula cribriformis. Two types of initial lymphatic ves- migrated into initial lymphatic vessels through mesothe- sels on the parietal peritoneum. Flat type (Left) and pro- lial stomata and holes of macula cribriformis truded type (Right). The former is found on the Morrison’s 12 Y. Yonemura et al.

OMS are found on the omentum and are the macula cribriformis is detected (Fig. 1.12). Under small lymphatic organs for the migration of peri- whole-mount preparation specimens stained with toneal inflammatory cells and the absorption of 5′-nucleotidase (5′-Nase) and alkali-phosphatase peritoneal fluid. Mean number of OMS in adult is double enzyme staining, agglomerated blood 26/cm2 and the diameters range from 15 to capillaries are found under the macula cribrifor- 800 μm [2, 79]. OSM is also linked to the dis- mis of OMS (Fig. 1.11, upper left). Additionally, semination of cancer cells [80]. Under scanning 5′-Nase-enzyme-staining shows a lymphatic electron microscopy, OMS was found to have plexus under macula cribriformis of OMS oval or round concave structure covered with (Fig. 1.13). Figure 1.14 is a vertical section of cuboidal mesothelial cells (Fig. 1.11, upper human OMS, stained with D2-40 monoclonal right). After digestion of cuboidal mesothelial antibody (Mab). Lymphatic plexus stained brown cells by 6 N KOH, concave pouch with holes of with D2-40 Mab is found beneath the cuboidal mesothelial cells. Surface of OMS small gaps between cuboidal mesothelial cells similar to stomata on the diaphragm are found (Fig. 1.15), and macrophages are detected in the mesothelial gap (Fig. 1.16). Intraperitoneal inflammatory cells and PFCCs migrate into OMS lymphatic vessels through the mesothelial cell gap from peritoneal cavity to OSM lymphatic plexus [80, 81] (Figs. 1.11–1.16). These findings are similar to the mechanisms of the leukocyte extravasation into the inflammatory stroma 18[ ]. These results indicate that lymphatic plexus of OMS can be considered a kind of initial lymphatic vessels. Structure of initial lymphatic vessels outside Fig. 1.9 Stomata on the diaphragm. Gap between meso- OMS is different. Figure 1.17 shows the loca- thelial cells of diaphragm, which connects with submeso- tions of submesothelial lymphatic vessels and thelial lymphatic vessel through holes of macula blood vessels of human parietal peritoneum. The cribriformis, located just below the mesothelial basement part of the lymphatic vessels in the parietal peri- membrane

Fig. 1.10 Holes of submesothelial basement membrane (Left). Macula cribriformis below basement membrane after 6N KOH cell maceration treatment. Diameters of the holes range from 5 to 30 μm (human peritoneum, Right) 1 Mechanisms of Peritoneal Metastasis Formation 13

5’-Nase-ALPase ALPase-positive arterial blood OMS capillaries (blue stain)

Peritoneal cavity

MS: Mesothelial stomata Initial lymphatics (IL) Flatter Cuboidal On removal Macula Submesothelial mesothelial mesothelial of OMS cell cribriformis-like collagen cells cells elements foramens plate

OMS Omental foramen SMCP

IL Ly Ly Ly Ly IL IL BV BV BV CP Ly

OMS

Cuboidal Submesothelial CP: glomerular mesothelial collagen arterial capillary cells plate (SMCP)

Fig. 1.11 Omental milky spot stained with 5′-nucleotid- concave, and the cuboidal mesothelial cells cover the ase and alkali-phosphatase double enzyme staining basement membrane of the bottom. Lymphatic plexus (Upper left), electron microscopic finding (Upper right), (red) is found below the macula cribriformis and the schema of the structure. OMS is oval or round toneum that are attached to macula cribriformis vessels alone are considered to communicate and mesothelial stomata are called initial lym- with peritoneal cavity. The size of the holes of phatic vessels (Fig. 1.8). There are two types of macula cribriformis ranges from 5 to 30 μm. initial lymphatic vessels, i.e., flat type (Fig. 1.8, PFCCs migrate into the initial lymphatic vessels left, and Fig. 1.18) and protruded type (Fig. 1.8, through the stomata on the mesothelial surface right, and Fig. 1.19). The former type is found on without destruction of macula cribriformis the Morrison’s pouch, paracolic gutter, and small (Fig. 1.20) and then proliferate in the lymphatic bowel mesentery. After intraperitoneal injection vessels (Fig. 1.21). of activated carbon CH40 [82], the tip of flat type The triplet structure consisting of mesothelial of initial lymphatic vessel alone is stained with stomata, hole of macula cribriformis, and initial CH40. The blind-looped lymphatic vessels lymphatic vessels is essential for the migration of extending from the submesothelial lymphatic PFCCs into the submesothelial lymphatic vessels vessels are the protruded type, and their blind tips (Fig. 1.22). When the stomata, hole of macula attach to the holes of macula cribriformis and sto- cribriformis, and the tip of initial lymphatic ves- mata (Fig. 1.8, right, Fig. 1.19). Since there is no sels are aligned in a row, direct communication adhesion of CH40 except at the tip of initial between peritoneal cavity and submesothelial lymphatic vessel, the tips of initial lymphatic lymphatic vessels is established, resulting in the 14 Y. Yonemura et al.

Fig. 1.12 The bottom structure of OMS after digestion cluster below the OMS basement membrane. Below the by 6 N KOH. Basement membrane is found beneath the holes of macula cribriformis, lymphatic and vascular cuboidal mesothelial cells. Holes of macula cribriformis plexus are found (Fig. 1.11, upper left, Fig. 1.13)

diaphragmatic initial lymphatic vessels drains to the deep-seated lymphatic vessels of diaphragmatic muscle and then flows to the para-aortic lymph nodes via collecting lymphatic vessels in triangular ligaments or along subdiaphragmatic arteries, and to the lymphatic vessels along the internal mammary artery (Figs. 1.23 and 1.24). PFCCs are adsorbed on the stomata by negative pressure of inspiration and migrate into the diaphragmatic initial lymphatic vessels. Figure 1.25 shows the metastasis from colorectal cancer in the diaphragmatic lymphatic vessel. Triplet structures (Fig. 1.22) are detected on Fig. 1.13 Lymphatic plexus locate under the macula the parietal peritoneum except on the anterior cribriformis of OMS (5′-nucleotidase enzyme staining, omentum of Japanese monkey) upper abdominal wall. The peritoneum of diaphragm, pelvis, paracolic gutter, Morrison’s pouch, and perihepatic ligaments does not have migration of PFCCs into the initial lymphatic any milky spots, but it does have the triplet struc- vessels. ture. In the experimental study, intraperitoneal Lymphatic system of diaphragm is different inoculation of cancer cells induces mesothelial from that of other parietal peritonea. Many lym- cell contraction (Fig. 1.4), and cancer cells were phatic stomata (Fig. 1.9) and plenty of submeso- detected in the submesothelial lymphatic vessels thelial lymphatic plexuses are detected by on day 3 after intraperitoneal inoculation [80]. 5′-Nase enzyme staining (Fig. 1.23). Lymphatic On the small bowel mesentery 2 cm in from the fluid adsorbed from peritoneal cavity through attachment to small bowel, many milky spot-like 1 Mechanisms of Peritoneal Metastasis Formation 15

Fig. 1.14 Human OMS stained with D2-40 Macrophagex with activated carbon and cuboidal mesothelial cells monoclonal antibody. Lymphatic plexus Flat mesothelial cells (brown) are found beneath the cuboidal mesothelial cells

Lymphatic plexus of OMS

Fig. 1.15 SEM findings of the surface of human Fig. 1.16 Macrophage is found in the gap (mesothelial OMS. Gaps (stars) between cuboidal mesothelial cells, stoma) between cuboidal mesothelial cells and the gaps connect with macula cribriformis and initial lymphatic vessels

Fig. 1.17 Normal structure of human Mesothelial cells Initial lymphatic vessel (brown) Morrison’s pouch stained by D2-40 monoclonal antibody. BPB# Initial lymphatic vessels attached to mesothelial cell gap. Blood vessels locate in the deeper subperitoneal tissue than lymphatic vessels. BPB blood peritoneal barrier

Blood vessels 16 Y. Yonemura et al.

ジMacrophage (CH40)

Fig. 1.18 Flat type of initial lymphatic vessel of subperitoneal lymphatic plexus except for flat type initial Morrison’s pouch. Initial lymphatic vessel is stained black lymphatic vessel. Accordingly, the tip of lymphatic vessel with CH40 (activated carbon), introduced intraperitone- stained with CH-40 is considered to contact with perito- ally before sampling. There is no CH40 attachment on the neal cavity

Fig. 1.19 Protruded carbon particles type of initial lymphatic vessel found in pelvic peritoneum. Tip of initial lymphatic vessel is stained with CH40, introduced intraperitoneally before sampling. There is no adsorption of CH40 except at the tip of initial lymphatic vessel (Left). CH40 particles adhere on the junction between lymphatic mesothelial cells

Fig. 1.20 SEM findings of peritoneal free cancer cells from gastric cancer migrate into initial lymphatic vessels through the holes of macula cribriformis 1 Mechanisms of Peritoneal Metastasis Formation 17

Initial lymphatic vessel Initial lymphatic vessel

リンパ管

Fig. 1.21 Findings of immunohistochemical staining liferate in the flat type (Left) and protruded type of initial using D2-40 monoclonal antibody for pelvic peritoneum lymphatic vessels (Right) from patients with gastric cancer. Gastric cancer cells pro-

Fig. 1.22 Schema of stomata, macula cribriformis, and initial Mesothelial layer lymphatic vessels. When the stomata, hole of Mesothelial stomata macula cribriformis, and the tip of initial Macula cribriformis lymphatic vessels are aligned in a row, direct communication between peritoneal cavity and Colagen fiber plate submesothelial lymphatic vessels is Lymphatic anchorring established, resulting in filament Lymphatic vessel the migration of PFCCs into the initial lymphatic vessels

Right diaphragm Right diaphragm

Anterior abdominal Anterior abdominal wall Wall*

Morrison’s pouch

Fig. 1.23 Metastatic nodules are found on the right dia- (Right). Lymphatic plexus is scarce on anterior abdominal phragm and Morrison’s pouch, but are not detected on the wall, but plenty of lymphatic plexuses are detected on the anterior abdominal wall (Left, star). Diaphragmatic lym- subdiaphragmatic surface phatic network stained with 5′-Nase enzyme staining 18 Y. Yonemura et al.

Mesothelial gap D2-40 staining Lymphatic/mesot Holes of macula Mesothelial layer helial stoma cribriformis Lymphatic Abdominal cavity Subperitoneal stomata initial lymphatics Subperitoneal LV LV collagen plate

Subperitoneal initial lymphatic vessel Diaphragmatic muscular layer

Subpleural collagen plate Diaphragmatic muscle

Pleural esothelial layer Subperitoneal initial lymphatic vessel

Fig. 1.24 Diaphragmatic lymphatic system. Left: mesothelial stomata. Right: Immunohistochemical stain- 5′-Nase enzyme staining shows the connection of holes of ing using D2-40 monoclonal antibody shows the lym- macula cribriformis and diaphragmatic initial lymphatic phatic stomata and diaphragmatic initial lymphatic vessels. Middle: Schema of diaphragmatic lymphatic sys- vessels. PFCCs are adsorbed through the stomata by nega- tem. Mesothelial gaps that do not connect with initial lym- tive pressure of inspiration and migrate into the diaphrag- phatic vessel are not called stomata, but those that matic initial lymphatics communicate with initial lymphatic vessels are named

that the metastasis in the area must be involved by trans-lymphatic metastasis (Fig. 1.26). Trans- lymphatic metastasis is found in gastric, colorectal, and pancreas cancer. However, lymphatic system in peritoneum covering the rectus abdominis muscle between hypochondrium and semilunar arc is quite different from that of other parts of peritoneal surface. In this area, no initial lymphatic vessels or Lymphatic submesothelial lymphatic plexuses are detected. vessels Lymphatic vessels locate in deep subperitoneal tissue 200 μm from the peritoneal surface Fig. 1.25 Lymphatic metastasis from colorectal cancer (Fig. 1.27), and the blood vessels are also scarce. in diaphragmatic lymphatic vessel (immunohistochemical Accordingly, trans-lymphatic metastasis does not staining using D2-40 monoclonal antibody) develop in the area. The peritoneal area must be involved at the late stage of PM and should be preserved when there is no macroscopic involve- structures are found. On this special peritoneal ment on the sector. area, round or oval shaped structures covered with cuboidal mesothelial cells are detected by SEM (Fig. 1.26). Below the cuboidal mesothelial cells, 1.4 Mechanisms of Superficial macula cribriformis is detected (Fig. 1.26, mid- Growing Metastasis dle). Since the area is frequently involved in PM, and CH40 injected into peritoneal cavity adheres PFCCs from appendiceal mucinous neoplasm on the area, absorption of CH40 by initial lym- (AMN) cannot metastasize through trans- phatic vessels is suggested. These results indicate mesenteric or trans-lymphatic metastasis, because 1 Mechanisms of Peritoneal Metastasis Formation 19

CH44

Fig. 1.26 Milky spot-like structure detected on the small CH40 injected into peritoneal cavity adheres on the peri- bowel mesentery in 2 cm from the attachment to small toneal area, suggesting absorption of CH40 by initial lym- bowel (Left). Below the cuboidal mesothelial cells, holes phatic vessels (Right) of macula cribriformis are detected by SEM (Middle).

200 m m

Fig. 1.27 Lymphatic vessels of anterior abdominal wall stained with D2-40 monoclonal antibody. Upper right: between hypochondrium and semilunar arc. Lymphatic Lymphatic vessels of falciform ligament, stained with vessels are located 200 μm from the peritoneal surface D2-40 monoclonal antibody. Lower left: Blood vessels of (Left). Lymphatic vessels and blood vessels in falciform anterior abdominal wall, stained with CD31 monoclonal ligament located just below the mesothelial cells (Right). antibody. Lower right: Blood vessels of falciform liga- Upper left: Lymphatic vessels of anterior abdominal wall ment, stained with CD31 monoclonal antibody

PFCCs of AMN are large and covered with muci- However, AMN can establish PM in depen- nous material (Fig. 1.28). They cannot migrate dent areas such as on the pelvis, subdiaphrag- into the submesothelial tissue or initial lymphatics matic surface, and greater omentum. They also (Fig. 1.29). grow in the pocket-like structure of omental 20 Y. Yonemura et al.

Fig. 1.28 Peritoneal free cancer cells of appendiceal cells show high proliferative activity (Right, mucinous neoplasm. Neoplastic cells are covered with Immunohistochemical staining using MIB-1 monoclo- mucinous material and the diameter is several hundred nal antibody) micrometers (Left, Alcian blue staining). Neoplastic

Fig. 1.29 Mechanism of superficial growing Adsorption of peritoneal free cancer cells metastasis. Peritoneal from appendiceal mucinous neoplasm free cancer cells from appendiceal mucinous neoplasm attach on the pelvic peritoneum by the interaction of mucinous material and adhesion Subperitoneal lymphatic vessel Blood-peritoneal Initial molecules (CD44) barrier lymphatic expressed on mesothelial (90 mm) vessel cells and/or by gravity

Blood vessel

bursa (inferior and superior recess of omental shows newly formed vasculature in the mucinous bursa), intersigmoid recesses, recesses in material without epithelial cells. These results duodeno-jejunal folds, and ileocecal fossa. strongly suggest that angiogenesis factors A large volume of mucinous materials with released from mucinous materials induce angio- tumor cells accumulates on the dependent perito- genesis in the mucinous stroma. neal parts as a result of peritoneal fluid resorption Figure 1.31 shows the metastasis on the sur- by the negative pressure of initial lymphatic ves- face of ovary by AMN. HE staining shows low- sels and/or gravity [79, 83]. grade mucinous neoplasm growing on the surface PFCCs of AMN attach on the pelvic perito- of ovary (Fig. 1.31, upper left). Angiogenesis neal surface by gravity or by the interaction of from the preexisting ovarian vasculature and epi- adhesion molecules on the mesothelial cells and thelial cells in the proliferating phase (positive mucinous materials. As shown in Fig. 1.30, muci- stain by MIB-1 antibody) are found. nous material attaches on the paravesical fossa, On the omental surface, PFCCs with muci- and immunohistochemical staining with CD31 nous materials from AMN are adsorbed on OMS, 1 Mechanisms of Peritoneal Metastasis Formation 21

Fig. 1.30 Mucinous material without epithelial cells Immunohistochemical staining using anti-CD31 mono- accumulates on paravesical fossa (Left). HE staining of clonal antibody shows newly formed vasculature (Right) the vertical section of the bar in left photograph (Middle).

Fig. 1.31 Superficial growing metastasis on ovarium from ovary (Upper right). Newly formed vasculatures from pre- appendiceal mucinous neoplasm (AMN) (Upper left). existing ovarian blood vessel are found (Immunohistological AMN growing on the surface of ovary with production of staining (HIS) using anti-CD31 Mab) (Left lower). Right mucinous material, and the neoplastic cells grow on the lower photograph shows proliferative activities of tumor ovarian surface showing pushing invasion into the corpus of cells (IHS using MIB-1 mAb) and many flat mucinous spots are found on the tum, and normal omentum (Fig. 1.32, middle, OMS (Fig. 1.32, left). HE staining shows three and Fig. 1.33). Inflammatory layer shows CD34- layers, consisting of a metastatic layer, inflamma- positive interstitial tissues. CD34 is a glycopro- tory layer between metastatic layer and omen- tein expressed on interstitial stem cells and