Progress on immuno-microenvironment and immune-related therapies in patients with pseudomyxoma peritonei

Pseudomyxoma peritonei (PMP) is an indolent malignant syndrome. The standard treatment for PMP is cytoreductive surgery combined with intraperitoneal hyperthermic chemotherapy (CRS + HIPEC). However, the high recurrence rate and latent clinical symptoms and signs are major obstacles to further improving clinical outcomes. Moreover, patients in advanced stages receive little benefit from CRS + HIPEC due to widespread intraperitoneal metastases. Another challenge in PMP treatment involves the progressive sclerosis of PMP cell-secreted mucus, which is often increased due to activating mutations in the gene coding for guanine nucleotide-binding protein alpha subunit (GNAS). Consequently, the development of other PMP therapies is urgently needed. Several immune-related therapies have shown promise, including the use of bacterium-derived non-specific immunogenic agents, radio-immunotherapeutic agents, and tumor cell-derived neoantigens, but a well-recognized immunotherapy has not been established. In this review the roles of GNAS mutations in the promotion of mucin secretion and disease development are discussed. In addition, the immunologic features of the PMP microenvironment and immune-associated treatments are discussed to summarize the current understanding of key features of the disease and to facilitate the development of immunotherapies.


Introduction: the epidemiology, pathologic process, and classification of pseudomyxoma peritonei (PMP)
PMP is a malignant tumor syndrome that is characterized by excessive mucus within the abdominal cavity and the accumulation and redistribution of mucous tumor cells 1 .Werth initially characterized PMP as a distinctive response of the peritoneum to a gelatinous substance produced by an ovarian neoplasm in 1884 2 .The association of PMP with an appendiceal mucocele was subsequently documented by Frankel in 1901 3 .The word "pseudomyxoma" is derived from the outdated term "pseudomucin" 4 .Researchers in the 18 th -19 th century believed that the extracted jelly material from the abdomen was distinct from the mucus secreted by digestive tract gland cells 5 .In recent decades the pathogenesis and treatment strategy for PMP have been a matter of controversy 6,7 , but the term, PMP, has been preserved and is well-recognized as a formal classification by cancer registries in the International Classification of Disease for Oncology (ICD-O) 8 .
Due to the rarity of PMP, global epidemiologic data is insufficient 9 .It has been suggested that among PMP patients in the Netherlands, the median age at diagnosis ranged between 57 and 62 years 10 .An epidemiologic survey of PMP conducted in Norway and England showed that the average incidence from 2009-2018 in both regions was 3.2 per million 11 .Another review indicated an incidence of 2 cases of PMP per 10,000 laparotomies, with women being 2-3 times more susceptible than men 12 .Considering 3.2 cases of PMP per million as the incidence of PMP and demographic data in China, we previously calculated that between 2010 and 2020, there were approximately 35,000 PMP patients and the prevalence was 25.1 cases of PMP per million in China 9 .Due to the lack of understanding of this rare disease, diagnoses are often delayed or even missed, thereby delaying optimal treatments.
The majority of the PMP cases originate from appendiceal mucinous tumors, while less common sources include mucinous tumors of the ovary, intestinal tract, urachus, and other intra-abdominal organs (Figure 1A).The classic pathologic process can be described as follows.First, appendiceal mucinous tumor cells proliferate and continuously produce mucus, blocking the appendiceal cavity and forming an appendiceal mucinous cyst.The pressure in the appendiceal cavity then increases and ultimately peaks, leading to perforation and rupture of the appendix.As a result, mucus-containing tumor cells are released into the abdominal cavity with spread to the peritoneum and abdominal organs.
Intra-abdominal fluid follows a specific pathway under physiologic conditions, mainly via the right paracolic groove, reaching the key reabsorption sites, including the right diaphragm and omental peritoneum.Subsequently, the fluid enters the bloodstream through peritoneal lymphatic holes and sub-peritoneal lymphatic vessels 13 .Importantly, the flow of intra-abdominal fluid is affected by gravity and tends to accumulate in the pelvic cavity.By way of analogy, mucus carrying the tumor cells resembles pirates sailing in motorless corsairs, passively drifting with the ocean current, and only disembarking when anchored at the harbor (Figure 1B).Therefore, the PMP cells and mucus congregate at the reabsorption sites within the peritoneum and pelvic cavity.These cells then colonize the peritoneal mesothelium and form visible tumor nodules, a phenomenon commonly referred to as "redistribution" (Figure 1C).
Massive amounts of mucus accumulate in the peritoneal cavity and progressively scleroses, leading to an increase in abdominal girth and intestinal obstruction with symptoms, such as abdominal pain and distension 14 (Figure 1D).In the terminal stage, tumor tissue and mucus fill the entire abdominal and pelvic cavities, impairing gastrointestinal motility.Patients find it difficult to ingest food or eliminate waste and ultimately succumb to severe cachexia 12 .
Due to the histopathologic heterogeneity of PMP the pathologic classification has been in chaos for decades until 2016, when the Peritoneal Surface Oncology Group International (PSOGI) published a well-known classification 8 .The pathologic grades of PMP include low-grade mucinous carcinoma peritonei (LMCP), which corresponds to disseminated peritoneal adenomucinosis (DPAM), high-grade mucinous carcinoma peritonei (HMCP), which corresponds to peritoneal mucinous carcinomatosis (PMCA), and high-grade mucinous carcinoma peritonei with signet ring cells (HMCP-S), which corresponds to peritoneal mucinous carcinomatosis with signet ring cells (PMCA-S 8 ; Table 1).
The term "acellular mucin", which indicates the presence of intraperitoneal mucin without identifiable neoplastic epithelial cells, was formerly controversial in the pathologic classification of PMP 16 .Acellular mucin has been reserved as a descriptive diagnosis and can be appropriate when the clinical manifestation of a patient is consistent with the PMP diagnosis (Figure 1E).LMCP is characterized by large mucin pools with a localized and sparse adeno-mucinous epithelium, the latter containing elongated columnar cells and few goblet-like cells, harboring mucin, and relatively consistent nuclei 6 (Figure 1F).The epithelial strips of LMCP on hyalinized stroma display inconspicuous atypia, apoptotic features, or mitotic activity 12 .Patients with acellular mucin or LMCP generally have a favorable prognosis.
The definition of HMCP depends on the presence of any factors that indicate high-grade cancer.Most features of HMCP-associated tumors are similar to LMCP, yet the former is characterized by marked cellular atypia and fusion of glandular structures (Figure 1G).In contrast, HMCP-S tumor cells exhibit conspicuous cellular atypia, invasiveness, and increased mitosis and apoptosis rates.The most characteristic feature of HMCP-S is signet ring cells, in which the nuclei are pushed aside by abundant mucin (Figure 1H).
The biological behavior of PMP cells often does not match the morphologic features 17 .For example, PMP tumor cells seldom metastasize to regional lymph nodes by lymphatic vessels or to distant organs by capillaries, and only weakly infiltrate adjuvant tissues.Although some of the PMP cells have a rather innocuous and less invasive appearance under the microscope, PMP cells spread throughout the peritoneum and secrete enormous amounts of mucin.In addition, relatively low-grade malignancies often relapse and aggravate PMP symptoms, resulting in a protracted disease course and eventually patient death 6 .The unique characteristics of PMP include massive mucus secretion and an extremely high recurrence rate that are independent of the pathologic grade.Conventional surgery, which refers to an en bloc resection of the primary tumor and adjacent tissues, does not achieve "radical tumor resection" because multiple lesions have formed and free PMP cells are floating and wandering in the peritoneal cavity.Cytoreductive surgery (CRS) and intraperitoneal hyperthermic perfusion (IPHP) was performed as early as 2003 on Italian patients with PMP in a multicentric phase II study 18 .The optimal cytoreductive rate was 87% with a 91% 5-year overall survival and 54% progression-free survival, which demonstrated acceptable feasibility of CRS + IPHP as the predecessor of CRS combined with hyperthermic intraperitoneal chemotherapy (HIPEC) 18 .Sugarbaker suggested CRS + HIPEC as the standard of care for epithelial appendiceal neoplasms and PMP in 2006, after which this treatment was gradually accepted and popularized by professional groups 19,20 .Visible tumors are removed by CRS, which consists of a peritonectomy and resection of tumor-affected organs, while HIPEC eradicates residual neoplastic cells at the microscopic level, which achieves a thoroughly curative effect 21 .By adopting this therapeutic strategy, Sugarbaker et al. reported survival rates of 71.9% and 54.5% at 5 and 10 years, respectively 22 .Based on the effective treatment of CRS + HIPEC and combined with peritoneal carcinomatosis (PC) studies conducted in China, we took the lead in formulating the first Chinese expert consensus of PC, including PMP 23 .
The professional diagnosis and treatment of PMP patients requires multidisciplinary teams in specialized peritoneal metastases (PM) treatment centers but there are acute shortages of such centers throughout the world 9 .In addition, patients with high-grade disease or widespread PM may not be candidates for surgery and are eligible for palliative therapy alone.Consequently, additional treatment options for PMP are needed, suggesting importance of further in-depth research.

The GNAS mutation and its MUC2promoting role in PMP
The predominant variants in PMP are Kirsten rat sarcoma viral oncogene homologue (encoded by KRAS) and guanine nucleotide-binding protein alpha subunit (encoded by GNAS), which exhibit an average mutation frequency of 66.7% and 49.5% for KRAS and GNAS, respectively 24 .The GNAS mutation is widely found in mucinous tumors, including mucinous tumors of the appendix and intraductal papillary mucinous neoplasms of the pancreas.Both of these tumors progress indolently but exhibit an excessive production of mucus 25 .In fact, a specific impact of the GNAS mutation on the promotion of mucin secretion has been identified 26 and the GNAS mutation correlates closely with excessive mucin production.The cAMP levels have been shown to be elevated and MUC2 and MUC5AC are both overexpressed in cells expressing an allele of GNAS which leads to an R201H mutation in the protein.The altered gene expression has been shown pharmacologically to depend on protein kinase A (PKA) activity 26 .
The GNAS gene (also referred to the GNAS complex locus) encodes an α subunit of the stimulatory heterotrimeric G protein (Gsα) and is situated on chromosome 20q13.32,at specific genomic coordinates (57,414,773-57,486,247)  25 .Commonly identified mutations involve Chr 20: 57484420: c.601 C > T or Chr 20: 57484421: c.602 G > A variations (Figure 2A).These changes lead to the substitution of Cys or His, respectively, for Arg201 in the protein (Figure 2B).Amino acid 201 is adjacent to the GTP binding site in the three-dimensional structure of the Gsα subunit, as illustrated in Figure 2C.The mutations lead to a structural alteration in the GTPase domain and the mutant Gsα proteins are unable to hydrolyze GTP, which results in a constitutively active status.These mutant proteins thus continually transmit signals to adenylyl cyclase (AC), essentially independent of the activation of a cognate G protein-coupled receptor (GPCR), to ultimately increase intracellular cAMP (Figure 2D).The resulting activation of PKA-related signaling pathways then modulates mucin production 27,28 .The mechanism likely involves the PKA-dependent activation of cAMPresponse element-binding protein (CREB), which forms a complex with proteins of the activating transcription factor (ATF) family 29 , translocates to the nucleus, and activates transcription of mucin-related genes.
Among the primary gel-forming mucins in PMP are mucin 2 (MUC2), mucin 5B (MUC5B), and mucin 5AC (MUC5AC).Specifically, MUC2 stands out for its relative abundance in mucus and the universally high detection in immunohistochemical analyses 30,31 .As discussed in our previous review, gel-forming mucin MUC2 exhibits the highest frequency of expression in PMP, with positivity observed in 99.1% (314/317) of analyzed specimens 25 .Because of the importance of the secretion of this protein in the induction of mucin secretion by the GNAS mutations, the mechanism connecting Gsα mutations to MUC2 overproduction is described to illustrate the general process (Figure 2).
The MUC2 gene, which codes for MUC2, is located on chromosome 11p15.5 32.The promoter of this gene contains a cAMP response element (CRE) that responds to activated CREB to increase transcription of the MUC2 gene 33,34 (Figure 2E).

The initial site of synthesis of the mucin monomer (apomucin) is ribosomes on the rough endoplasmic reticulum (RER).
Nascent apomucin is transferred into the ER with a ribosomal translocon complex 35 , where N-acetylglucosamine (GlcNac) is added to Asn residues 36 (Figure 2F).The N-glycosylated apomucin is then modified by the removal of Glc residues and dimerized through disulfide bond formation.After transport from the ER into the Golgi apparatus, the apomucin is  O-glycosylated on repeating Pro, Thr, Ser (PTS) sequences 37 (Figure 2F).The modified mucin is later multimerized to a condensed state, which buds off from the trans-Golgi network into vesicles and forms the mature secretory granules for storage and secretion by exocytosis 38 (Figure 2G).After the release of mucus granules into the intestinal lumen, MUC2 polymers undergo a significant size increase, expanding by > 1,000-fold due to depolymerization and hydration 39 .This expansion leads to the unfolding of MUC2 polymers and formation of an extensive reticular lamellar structure 40 (Figure 3).
As the primary causes of mortality in PMP patients, intestinal obstruction and organ adhesion are induced by progressive mucinous sclerosis, which are derived from the massive accumulation as well as changes in the physical and chemical properties of mucin, and are referred to as mucus viscoelasticity 41 .The initial state of mucus is colloidal but eventually transforms into a semi-solid then solid state.Consequently, progressive mucinous sclerosis and changes to mucus viscoelasticity stand as the central pathologic mechanisms driving the development of PMP.These changes to the mucus viscoelasticity can be explained as follows 31 .The turbidity and kinematic viscosity of mucus, which reflects the physical characteristics, have been shown to increase > 270% and 40%, respectively.Moreover, the sialic acid content of the mucus increases by > 230%, meaning that the relative amount of protein decreases.Third and specifically regarding the most abundant mucin (MUC2), between 80% and 90% of the dry weight comes from oligosaccharide side chains, and the protein itself is rich in Thr and Pro residues, which increases the viscosity further 42 .Finally, the relative amounts of the various mucins within the material contributing to the viscoelasticity.Specifically, the MUC2:MUC5B:MUC5AC ratios are 2.25:1.54:1.0,1:1:1, and 3:2:1 in soft, semi-hard, and hard mucus, respectively 31 .The mucins form an insoluble mucous barrier that protects the gut lumen and respiratory tract.However, the mucins also participate in shaping the characteristic tumor microenvironment (TME) of PMP and influence the effectiveness of clinical treatments, including chemotherapy and immunotherapy.

Immunologic features of the PMP TME Overview of the tumor immune microenvironment (TIME)
The TME is a complicated network with heterogeneous components, including immune cells [T/B lymphocytes, tumorassociated macrophages (TAMs), and natural killer (NK) cells], stromal cells [tumor-associated fibroblasts (TAFs) and mesenchymal stromal cells], and non-cellular components (extracellular matrix, cytokines, and chemokines) 43 .Tumor cells can survive and multiply in large part due to the suppressive nature of the immunologic aspect of the TME (the TIME) 44 .For example, CD8 + cytotoxic T lymphocytes (CTLs) are normally a major contributor to the destruction of tumor cells but the anti-tumor effect of the TIME can impair CTL activity via multiple immunosuppressive factors, including immune checkpoint molecules and immunosuppressive cells 45 .Accordingly, immune checkpoint inhibitors (ICIs), which target programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), or other related molecules, have achieved remarkable efficacy in the treatment of multiple cancer types, such as melanoma and Hodgkin's lymphoma [46][47][48] .
Immunosuppressive cells, such as TAMs, regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSCs), function by being recruited to the tumor site where they produce immune-repressive cytokines and display immune checkpoint molecules to CTLs [49][50][51] .Targeting the TIME offers a considerable therapeutic benefit in contrast to directly focusing on tumor cells 52 because tumor cells tend to develop drug resistance owing to their genomic instability, whereas non-tumor cells within the TIME are less prone to mutation and thus less likely to develop resistance 52 .Unfortunately, the complexities of the components of the TIME and their interactions suggest that cells often play contradictory roles in tumor evolution.The typical example involves TAMs, which present as anti-tumor M1 or pro-tumor M2 phenotypes 53,54 .The future direction of immunotherapy in PMP may be directed at the contradictions and heterogeneity of its TIME.
In this review the limited information available is summarized and a model of the immunosuppressive environment of PMP is advanced, which is characterized by the existence of immunosuppressive cytokines and chemokines, increased infiltration of immunosuppressive cells, decreased expression of immune-effective cells, and upregulated immune-resistant markers expressed by tumor cells and within the TIME.

Immune cells in the PMP TIME
Various subsets of immune cells have the capacity to play supportive or inhibitory roles in tumor development and the orientations are influenced by environmental cues within the specific microenvironment they inhabit.Therefore, the impact of distinct immune cells infiltrating the TIME during PMP tumorigenesis need to be recognized because the immune cells are crucial for promoting an immune response against PMP.However, few studies have investigated immune cells in the TIME or their associations with tumor progression in PMP.
Scientists have observed an increased infiltration of immune cells, including CD68 + macrophages, CD3 + T cells, and CD20 + B cells, in the PMP TME, either in clustered or dispersed distributions 55 (Figure 3).The results of studies related to immune cells are summarized herein with a focus on depicting the overall picture of immune cell infiltration in PMP.

Hypoxia and TAMs
Hypoxia inducible factor-1α (HIF-1α) is quickly degraded by the ubiquitin-proteasome system in an oxygen-dependent degradation pathway and can only remain stable under hypoxic conditions 56 .Previous studies have identified the wide expression of HIF-1α in PMP cells 57,58 .Additional direct evidence comes from the work conducted by Valenzuela-Molina et al. 58 , who assessed the intratumoral real-time oxygen microtension (pO 2 mt) and oxidative stress marker (catalase) in the tumor-derived mucin of five PMP patients.Valenzuela-Molina et al. 58 identified significantly reduced pO 2 mt and catalase activity compared with the adjacent healthy tissue.Moreover, the expression of hypoxia-inducible CD39 and CD73 (as discussed in Section CD39, CD73 and adenosine A2A receptor) were detected in PMP cells and the surrounding TME 59 .These results suggest that the TME of PMP is hypoxic.
Inadequate vessel coverage and perfusion hamper not only oxygen delivery but also the transportation of immune cells and the accumulation of metabolic byproducts "fine-tune" the phenotypes of immune cells, including macrophages toward the M2 phenotype 60 (Figure 3).Kusamura et al. 59 showed that the expression of CD163 + M2 macrophages in the TME in 67% (25/37) of PMP cases analyzed and in the tumor cells in 27% (10/37) of the cases.In this hypoxic context, the M2 phenotype of TAMs emerged and promoted angiogenesis by releasing VEGFA and cathepsin and these cells also exert an immunosuppressive effect by expressing inhibitory ligands, such as PD-L1, and recruiting Tregs 61,62 .In addition, the presence of Gram-negative bacteria, such as Helicobacter pylori, has been identified in the PMP microenvironment 63 .Components of the bacterial cytoderm lipopolysaccharide aberrantly activate AMP-dependent protein kinase and hinder the progression of macrophages to the M1 phenotype 64 .It should be noted that despite this circumstantial evidence, a direct link between hypoxia and phenotype regulation of macrophage in PMP has not been confirmed.

Immunosuppression of MDSCs and tumor-associated neutrophils (TANs)
MDSCs include a heterogeneous population of myeloid cells composed of a spectrum of immature cells, including mononuclear/macrophages, granulocytes, and dendritic cells, typically demonstrating immunosuppressive capabilities and occurring in pathologic circumstances 65 .MDSCs inhibit the immune cells from performing normal innate and adaptive immune functions 66 .In a study involving 37 PMP patients, CD15 + granulocytic MDSCs were detected in 31 (83%) of the cases, indicating the possible pro-tumor activity of these cells 59 (Figure 3).
Another immunosuppressive factor that is closely associated with MDSCs is granulocyte-macrophage colony-stimulating factor (GM-CSF), which has been detected in PMP cells and the PMP microenvironment 59 (Figure 3).GM-CSF stimulates TAMs to express immunosuppressive ligands, such as PD-L1/L2, and to inhibit T cell-mediated immune function by inducing MDSCs to enter tumor tissues 67 .GM-CSF induces the production of MDSCs from the bone marrow in a dose-dependent manner 68 .However, the exact regulatory relationship between GM-CSF and MDSCs requires further investigation.
MDSCs express some surface molecules and intracellular proteins that suppress the immune function of other myeloid cells, such as TANs 69 .The heterogeneous TANs can be categorized into a tumor-inhibiting phenotype (N1 neutrophils) and a tumor-promoting phenotype (N2 neutrophils) 60 .The principal cytokine inducing the pro-tumor functions of N2 neutrophils is TGF-β 70 , which is enriched in the PMP microenvironment 59 .Moreover, the infiltration of neutrophils in the TME has been identified in PMP tissues 63 and multiple studies have determined that an elevated neutrophil-to-lymphocyte ratio (NLR) in peripheral blood is significantly associated with a poor prognosis in PMP patients [71][72][73] .An increased number of neutrophils in the circulatory system with local infiltration in the tumor suggests an inhibitory effect on immunity.The phenotypes of these cells in the TME of PMP remains to be elucidated.

NK and B/T cells
In a study involving the features of NK cells in patients with low-grade peritoneal carcinomatosis, the unique immature phenotype of CD56 dim NK cells was observed in the microenvironment of the peritoneal fluid.This phenotype features relatively low expression of killer cell immunoglobulin-like receptor, leukocyte Ig-like receptor-1, and CD57, and high expression of NK cell receptor A 74 (Figure 3).Moreover, the study also showed a significant reduction in the expression of key activating receptors on NK cells (NKp46, DNAM-1, NKp30, and CD16) 74 .Although the expression of activating and inhibitory receptors on NK cells were impaired, the function of NK cells might be depressed and eventually result in evasion of immune surveillance.High levels of CXC chemokine receptor type 4 (CXCR4) were demonstrated on NK cells in the peritoneal fluid from PMP patients.Indeed, this chemokine receptor is widely expressed in a variety of hematologic and solid tumors 74 .Activation of this receptor by the cognate ligand, CXCL12, promotes tumor cell proliferation, survival, and metastasis.Inhibition of CXCR4 results in enhanced T and NK cell infiltration with reduced recruitment of TAFs 75 .Another study reported that CD56 + NK cells were absent in the TME of PMP, which also supports the immunosuppressive nature of the microenvironment 59 .
Research associated with B/T cells in PMP is limited.Kusamura et al. determined that 86% cases of PMP tissue expressed CD4 + T cells, among which 44% exhibited strong CD4 + expression 59 .In contrast, only 27% of cases expressed CD8 + T cells, with 90% cases of the positive cases exhibiting low CD8 + expression (Figure 3).Furthermore, in spite of a relatively wide expression (67%) of CD20 + B cells in PMP tissue, 76% of the positive cases expressed low CD20 +59 (Figure 3).

Stromal cells in the TIME of PMP
Stromal components, such as TAFs, also participate in PMP progression (Figure 3).A bioinformatics study showed significantly elevated TAF scores in an extended dataset of PMP samples, indicating the potential predominance of TAFs 76 .The existence of TAFs was identified by diffuse staining of fibroblast-activating protein and high levels of fibroblast growth factor 2 (FGF2) in PMP samples 57 .Interestingly, human placental growth factor (PlGF), which is a pro-angiogenic factor, was not detectable in PMP samples probed with an anti-human PlGF antibody, whereas an anti-mouse PlGF antibody returned positive signals in PMP tissues of a patient-derived xenograft (PDX) mouse model, illustrating a role for stromal secretion and tumor enhancement 57 .

Capillaries and lymphatic vessels
While the precise involvement of TIME in PMP has yet to be elucidated, preliminary knowledge has provided some intriguing insight.PMP shares some common TIME properties with other tumors, such as increased capillaries, lymphatic vessels, cytokines, and chemokines 55,77 (Figure 3).In addition, positive CD34 and LYVE-1 staining in immunohistochemical analyses of PMP tissues has further illustrated vascular and lymphatic upregulation, which is beneficial for nutrient and oxygen delivery to tumor cells (Figure 3).Andersson et al. 57 reported that angiogenesis is a central characteristic in PMP and demonstrated the efficacy of anti-angiogenic treatments.Andersson et al. 57 analyzed tumor tissue from patients and PDX models and detected elevated pro-angiogenic factors, including vascular endothelial growth factor A (VEGFA), PlGF, FGF2, and soluble FMS-like tyrosine 1 (sflt1).Furthermore, proliferation of human umbilical cord vascular endothelial cells were induced by ex vivo PMP tissue samples, while the proliferative effect was inhibited by VEGF pathway inhibitors (bevacizumab and aflibercept) in a PDX model 57 .

Tumor-associated antigens (TAAs)
The expression of some TAAs was also detected in PMP, including pituitary tumor-transforming 1 (PTTG1) and squamous cell carcinoma antigen 1 (SCCA1) 55 .The former TAA enhances tumorigenesis by activation of AKT, a key regulator of epithelial-mesenchymal transition and maintenance of stem-like properties in breast cancer cells 78 .This pathway results in upregulation of the transcription factor, Snail, changing the morphology of tumor cells to improve migration and invasion characteristics 78 .

Cytokines and chemokines
Lohani et al. 77 showed that the levels of interleukin-6 (IL-6), IL-8, interferon gamma-induced protein 10 (IP-10), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1α (MIP-1α) in ascites were 200-fold higher compared to normal values (Figure 3).However, there was no correlation between the cytokine and chemokine levels in ascites and the corresponding levels in serum, which indicated a relatively confined and unique TIME for PMP cells 77,79 .It is possible that the gelatinous ascites, which is rich in mucin, or the scarring and fibrosis surrounding the focus on the peritoneum restricts the diffusion of these small proteins to blood.While the reason for the confined contents remains unknown, the available evidence supports the peritoneal synthesis of these proteins by the tumor cells or associated stromal cells 77 .In addition, the localized distribution of some cytokines and chemokines were also demonstrated by immunohistochemistry, including IL-6 localizing in the stroma surrounding myoepithelial cells, IL-8 in adipose tissue, and IP-10 and MCP-1 in PMP cells 77 (Figure 3).This phenomenon reveals that the different components in TIME might have specific and distinct effects on PMP cells, whether in active secretions or through passive regulation.In addition, IL-6 within ascites has exhibited multiple associations with other cytokines and chemokines and appears to serve as a pivotal cytokine in the regulation of PMP 77 .A feedback loop involving IL-6R/STAT3/miR-34a is known to foster invasion and metastasis through EMT induction in colorectal cancer 80 .However, the biological significance of IL-6 for PMP requires further investigation.
Another important cytokine in PMP is transforming growth factor-β (TGF-β).TGF-β has central roles in early embryonic development and immune surveillance, and has been deemed an anti-cancer factor for the inhibitory effect on the proliferation of tumor cells 81 .Conversely, TGF-β also promotes growth during late tumor stages by mediating genome instability, EMT, and immune evasion.When the level of TGF-β increases, the differentiation of immature T cells into Th1 cells is blocked and the transformation into Treg subsets is promoted 82 .In addition, TGF-β also inhibits the antigen presentation function of dendritic cells and thus leads to immune evasion of tumor cells.In a study involving five patients with low-grade peritoneal carcinomatosis, which included two mesothelioma and three low-grade PMP cases, a high concentration of TGF-β1 was detected in the peritoneal fluid 74 (Figure 3).Although the study included a small number of PMP patients and did not exclude the interference of mesothelioma, the study emphasized upregulation of TGFβ and the underlying immunosuppressive effect on PMP.
Multiple studies have concluded that TGF-β is involved in the aberrant expression of receptors on NK cells, including a reduction in NKp30 and induction of NKG2A, together with CD16 impairment 83,84 .In contrast, the TGF-β signaling pathway appears to be repressed according to other studies that will be discussed below.

CD39, CD73, and adenosine A 2A receptor
CD39, also known as exonucleoside triphosphate diphosphate hydrolase 1, is a transmembrane protein in which the active center is on the extracellular domain 85 .CD73, also referred to ecto-5′-nucleotidase, exists on the cell surface in the form of homodimers or extracellularly in a free form 86 .The two proteins are ectonucleotidases.CD39 is capable of hydrolyzing extracellular ATP (eATP) to produce AMP, while CD73 hydrolyzes AMP into adenosine 87 .Subsequently, adenosine activates purinergic type 1 GPCRs receptors, which activate downstream pathways, such as the AC-cAMP-PKA pathway 88 .
One such adenosine GPCRs is adenosine A 2A receptor (A2AR), which is expressed on a series of immune cells and exerts immunosuppressive functions 60 .For example, the activation of A2AR by adenosine has been shown to promote macrophages into presenting an M2-like phenotype and to facilitate expression of PD-L2 and IL-10 in dendritic cells 89 .In addition, CD39 and CD73 facilitate Treg cells rapidly accumulation of adenosine by quick dephosphorylation, then promote the expression of CTLA4, PD-1, and Foxp3 to suppress local immunity 90 .
Positive expression of CD39, CD73, and A2AR has been identified in PMP cells and the TME, and intense and wide (80%) A2AR expression was observed in PMP cells 59 (Figure 3).Moreover, the expression of A2AR was shown to be independent of GNAS or KRAS mutational status, which suggests that aberrant A2AR activation is sufficient to stimulate the relevant pathways, even in the absence of constitutively-active Gsα.The hypoxic microenvironment of PMP can also contribute to the upregulation of CD39 and CD73, which is conducive to the accumulation of the immunosuppressive factor adenosine 60 .

COX-2, PGE2
Cyclooxygenase-2 (COX-2) is frequently upregulated in various cancers and is strongly linked to immunosuppression 91 .COX-2 catalyzes the production of prostaglandin E2 (PGE2), a bioactive lipid metabolite which contributes to the reprogramming of immune cells 92,93 .Excessive production of PGE2 fosters the growth and differentiation of Treg cells and M2-like TAMs, and also impedes the recruitment of conventional type I DCs by NK cells, which obstructs T cell infiltration 88 .Additionally, PGE2 suppresses the survival and function of CTLs, thereby facilitating tumor immune evasion 88 .
The expression of COX-2 in PMP cells has been reported but studies quantifying PGE2 levels in PMP are not available 94 (Figure 3).In consideration of the linkage between COX-2 and PGE2, COX-2 inhibitors, such as celecoxib, which reduce the production of PGE2, represent potential immune-related treatments.Indeed, the combination of a COX-2 inhibitor with anti-PD-1 therapy has been studied in an animal model, which provided a synergistic anti-tumor immune response 95 .Furthermore, a COX-2 inhibitor was shown to be capable of inhibiting mucin production in a murine xenograft model of PMP, although this mechanism does not fit within the scope of immunotherapy 96 .

Immune-related therapy in PMP
Since Spratt et al. 97 first presented the CRS + HIPEC method in 1980 this treatment has eventually become the standard of care for PMP and other peritoneal carcinomatoses.The safety and therapeutic effect of this strategy has been evaluated and confirmed 98 .In a multi-institutional registry study including 2,298 patients with PMP from 16 specialized units, the median survival and progression-free survival rates of patients who underwent CRS + HIPEC were 196 and 98 months, respectively 99 .
However, the effectiveness of CRS + HIPEC in individual patients can be influenced by multiple factors.For example, the 5-year survival rates in advanced-stage patients who achieved completeness of cytoreduction (CCR) level 2 or CCR3 was only 24%; the low survival mainly results from the gross residue 99 .In addition, the successful implementation of CRS + HIPEC requires experienced surgeons in specialized PMP treatment centers and the scarcity of qualified clinicians restrains the promotion of the treatment 9 .Another issue pertaining to treatments for patients with non-resectable and recurrent disease are currently not available.Therefore, developing a novel therapeutic strategy, such as immunotherapy, as an indispensable complement to CRS + HIPEC is urgent.
Currently, there is only one clinical trial which is associated with PMP immunotherapy [Dual Anti-CTLA-4 and Anti-PD-1 Blockade in Rare Tumors (DART, ClinicalTrials.gov ID: NCT02834013); Figure 4].Patients with low-grade mucinous adenocarcinoma of the appendix and ovary, including PMP, who met the inclusion criteria were eligible for the phase II clinical trial.Subsequently, patients without PD-L1 amplification were allocated to arm I and received nivolumab [anti-PD-1 (240 mg)] combined with ipilimumab [anti-CTLA-4 (1 mg/kg)] intravenously in a 42-day cycle.The other patients with PD-L1 amplification were then allocated to arm II and received nivolumab [anti-PD-1 (240 mg)] only (Figure 4).The primary objective was overall response rate (ORR) and the secondary objectives were overall survival (OS), progression-free survival (PFS), and clinical benefit rate.The clinical trial has been ongoing since 2016-07 and remains active until 2026-05.Until now, there are several publications about the study results, such as unresectable or metastatic metaplastic breast cancer 100 , advanced gallbladder cancer 101 , angiosarcoma 102 , and neuroendocrine neoplasm 103 .However, the PMP-associated results have not been reported.
Studies investigating immune-associated treatments for PMP are presented below.

Immunopotentiation
OK-432 (picibanil) is a lyophilized preparation made of low-toxicity Streptococcus pyogenes (group A) after treatment with penicillin, of which the main active ingredient is a non-specific immunopotentiator [OK-PSA (a lipoteichoic acid-related molecule)] 104 .OK-432 enhances the cytotoxicity of NK cells and lymphocytes, and promotes the secretion of some chemokines and cytokines, including interferon, IL-2, and TNF-α, which enhances the anti-tumor immune response 105 .Importantly, OK-432 has a stronger ability to promote the maturation of immature DCs compared with other maturation inducers 105 .
Fukuma et al. 106 delivered adjunctive immunotherapies with OK-432 to three patients with ovary-derived PMP after surgery (Figure 5).The immunotherapy commenced with IP administration of OK-432 during laparotomy, with doses ranging from 5-10 Klinische Einheit (KE).Subsequently, a sustained and regular intramuscular administration was maintained at doses ranging from 0.2-2.0KE twice per week.No apparent side effects were reported.Two patients received 11 and 16 months of intramuscular OK-432 injections, respectively.Remarkably, both patients did not relapse 7 and 6 years after the respective surgeries.The third patient underwent treatment with OK-432 as of the study publication and there was no indication of recurrence 4 months after surgery.The researchers also assessed immune competence by examining delayed hypersensitivity responses to skin-test antigens.Within 8 weeks of initiating OK-432 immunotherapy, all initially negative skin tests turned positive, demonstrating a notable enhancement in the immunologic status throughout therapy.The study supported the immune-promoting and anti-tumor effect of OK-432 that had previously been reported 107 .However, the number of patients receiving immunotherapy was low and the detailed mechanism by which OK-432 affects the immune system was not thoroughly investigated.
Apart from the direct suppression of tumor cells, OK-432 has the advantage of strong antigenicity, which can be used to support the effects of tumor vaccines.Li et al. 108 developed a tumor vaccine that coupled OK-432 to squamous carcinoma cells (KLN-205) and immunized a mouse tongue cancer model.The findings indicated that OK-432-conjugated KLN-205 vaccines triggered cytolytic activity, effectively suppressing the incidence and growth of KLN-205 tumors and enhancing survival of the mice.Additionally, histologic examination revealed an enhanced infiltration of lymphocytes around tumor cells after tumor inoculation in the vaccine group.These findings imply that tumor vaccines that include immunomodulators, such as OK-432, induces tumor-specific immunity, which is a feasible path towards immunotherapy.Tumor vaccines are discussed further in Section Tumor neoantigen vaccines.

Radioimmunotherapy (RIT)
RIT refers to the combination of radiotherapy and immunotherapy.Conjugated monoclonal antibodies (mAbs) that target the antigens on tumor cells are paired with a radioactive nuclide and are injected into the circulation or body cavities, delivering high doses of radiation directly to tumor cells 109 .Because the nuclide, such as 125 I or 131 I, is conjugated to a specific targeting antibody, the nuclide continuously irradiates the tumor cells internally at a low dose rate by emitting α-rays, β-rays, or Auger electrons, causing damage to DNA and organelles of tumor cells 110 .
Kairemo et al. [111][112][113] conducted a series of studies involving RIT by administering 131 I-labelled B72.3 mAb IP and intravenously (IV) to PMP patients (Figure 5).The target of mAb B72.3 is tumor-associated glycoprotein (TAG)-72, also known as carbohydrate antigen 72-4 (CA72-4), a pleomorphic epithelial mucin that is highly expressed in human adenocarcinomas, such as colon cancer, while the expression in normal tissues is very low 114 .Kairemo et al. [111][112][113] treated PMP patients with a low dose [90-120 megabecquerels (MBq)] of 131 I-labelled B72.3 by IV (2 patients) or IP (7 patients) injections and calculated the dosimetry and biodistribution 111 .The immunohistochemistry and digital autoradiography showed that the radiative uptake was proportionate to the quantity of targeted mAb on PMP cells.Moreover, gamma imaging indicated that the radiative uptake became enriched in the peritoneal cavity over time.These results indicated that 131 I-labelled B72.3 mAb is capable of specifically targeting the tumor.In addition, the IP dose of radiative accumulation was estimated to be 13 MBq by 3-dimensional quantification and most of the radioactivity remained in the abdominal cavity until 16 days after administration.No adverse reactions were observed when the antibody was administered and there were no false-positive uptake events recorded, indicating that the immunoradioactive mAb has good tolerance and targeting properties.
Nevertheless, some negative aspects of the treatment as revealed by the study are worthy of discussion.First, the distribution of the radioactivity of the 131 I-B72.3 mAb varied widely in different patients.In TAG-72-negative tissues, there was no radiative uptake detected.The researchers also reported uptake of 131 I in normal tissues of the thyroid, stomach, and salivary glands, which should be considered during clinical application.Another cautionary issue regarding the study is that the dose of radioactive mAb delivered IV was 1 MBq, a dose which was enough for imaging but not for therapeutic treatment.In two subsequent studies, a therapeutic dose (3-4 GBq) of 131 I-B72.3 mAb was administered to PMP patients 112,113 (Figure 5).However, there were no description of whether the clinical manifestation or prognosis of PMP patients were improved.
The procedure with which RIT is implemented has also been investigated.Boudousq et al. modified the format of HIPEC and conducted a "brief IP RIT" therapy in a mouse model of peritoneal carcinomatosis 115 .The process of brief IP RIT involves the IP injection of mice with 5 mL of an 125 I-labelled IgG1k 35A7 mAb, which targets CEA, or a saline control for 1 h, then flushed the peritoneal cavity for 15 min.The perfusion system was similar to that used for typical HIPEC in that the system includes inflow and outflow catheters as a liquid flow path and a peristaltic pump as the driving force.The study reported a low hematologic toxicity of brief IP RIT in mice and the radiation dose delivered to healthy organs (< 1 Gray) was significantly lower compared to IV RIT.Additionally, while the tumoral irradiation dose of IV RIT was higher than that achieved with brief IP RIT, the tumor-to-blood irradiation dose ratios were 1.7 and 5, respectively.These findings suggest that brief IP RIT offers protection to healthy tissues while still delivering substantial radiation doses to tumors.Finally, the median survival was highest in the group of mice treated with brief IP RIT combined with IV RIT (73 d) compared to control mice (32 d) and mice treated with brief IP RIT alone (46 d).The prognostic improvement of mice indicated a complementary tumor killing effect of the two RIT approaches.
Novel RIT techniques have recently emerged in studies of PMP and peritoneal carcinomatosis treatment, including bio-orthogonal click chemistry 116 .At present, RIT is mainly used to treat blood system-related tumors, but the therapeutic effect on solid tumors has not been satisfactory.The reasons for the lower effect on solid tumors may include poor tissue perfusion of solid tumors, resulting in poor anti-tumor efficacy, and the tendency of tumor cells to develop radiotherapy resistance 117 .In addition, IV RIT exerts hematologic toxicity due to its long blood circulation time and the possibility of inducing hematologic malignancies 118,119 .It should also be emphasized that RIT relies on highly specific targeted antibodies.Therefore, mAbs need to be tested for reactivity to PMP tissues prior to application.

Tumor neoantigen vaccines
The genetic instability of tumor cells frequently results in the accumulation of numerous mutations, leading to the generation of unique tumor-specific antigens, which are known as neoantigens.Therefore, the idea of tumor neoantigen vaccines that enhance immune memory response has arisen as a monoclonal antibody technique emerging in recent years 120 .A major problem encountered with immunotherapy is that the immunogenicity of relevant neoantigens might be too weak to elicit a useful response.In addition, the neoantigens are sometimes proteins, such as α-fetoprotein (AFP), that are normally expressed during development, and the immune system does not respond to such proteins.The combination of strategies based on neoantigen vaccines with either conventional therapies, such as chemotherapy or radiotherapy, or another immunotherapy, such as anti-PD-L1 therapy, might be the solution to the current challenges facing this novel treatment method.
The formulations of tumor neoantigen vaccines can include tumor lysates, tumor antigens or APC-presenting tumor lysates, combined with other types of vaccines, including nucleic acid vaccines, DC vaccines, and tumor polypeptide vaccines 60,121 .Few studies of the application of tumor neoantigen vaccines in PMP exist and the existing studies mainly focus on tumor polypeptide vaccines 122,123 (Figure 5).Graham et al. 122 immunized five PMP patients once or twice intradermally in the anterior thighs with a vaccine that contained autogenous mucin, Freund's adjuvant, killed Mycobacterium tuberculosis, mineral oil, and Arlacel A. The symptoms, signs, and prognostic information of patients after vaccination were recorded.Among the 5 patients, 3 received a single injection of vaccine and these patients survived 3.5 years, 2 years, and 3 months.No detailed information was available concerning other reactions to the mucin vaccine.Two of the patients were immunized twice and the patients did not report any physical discomfort.These two patients also had no evidence of recurrence 1 and 9 years after immunization.Thus, multiple mucin immunizations are apparently more effective than a single injection.However, the associated report included only a brief description of the strategy and outcomes, and no statistical information was provided.
Flatmark et al. 123 chose synthetic peptides containing Gsα mutations (R201H or R201C) as potential tumor neoantigen vaccines.Flatmark et al. 123 stimulated peripheral blood T cells from PMP patients and healthy donors with the vaccine and examined the reactions of T cells.Flatmark et al. 123 observed significant proliferative responses of PMP patient T cells stimulated with the mutant Gsα peptides as compared to cells stimulated with peptides with the wild-type (WT) sequence, suggesting the presence of a pre-existing immune response against mutant Gsα.The interferon-γ production of T cells showed significant responses against the R201H subtype, but not R201C, as compared to the wild-type.Additionally, in comparison to PMP patients, the magnitude of T cell responses to mutant Gsα in healthy donors was generally lower, which confirmed the immunogenicity of the mutant Gsα sequence because T cells from healthy donors would not likely have been pre-immunized by mutant Gsα.The researchers also detected the upregulation of immune checkpoint molecules (principally PD-1 and TIGIT) and exhaustion markers (CD69) on T cells by mass cytometry, suggesting that these T cells may be antigen experienced and tumor reactive.
Although mutant Gsα triggers a spontaneous immune response in PMP patients, the anti-tumor efficacy is inadequate as a tumor neoantigen vaccine for application.Therefore, Flatmark et al. put forward a therapeutic hypothesis ("pseudovax") in which an existing response and clonal expansion of T cells would be amplified by mutant Gsα.Afterwards, ICIs could be applied to strengthen the immunotherapy 123 .Recently a new peptide mixture (patent number: WO2022238571-A1) was registered in the Derwent Innovations Index by Eriksen et al. 124 .Composed of a peptide with a specific amino acid sequence, a T-cell mixture or pharmaceutical composition, this mixture is designed for the use as a vaccine or medication for PMP patients with GNAS mutation.The peptide mixture includes at least one ICI (an anti-PD-1/PD-L1 inhibitor is preferred) and enables simultaneous, separate, or sequential administration with the ICI 124 .This peptide mixture has not been widely applied or studied in the context of PMP but it represents a promising tumor neoantigen vaccine strategy for combinational or complementary treatment.

Future challenges and conclusions
Developments in basic research technologies, including next-generation sequencing, have led to an in-depth study of gene mutation profiles in PMP, leading to important advances 24,125 .However, PMP has a unique TME that contains excess gelatinous mucus and sparse tumor cells, which increases the difficulty of cell extraction and subsequent oncologic research.In addition, although successes of research involving cultured PMP cells and PDX animal models were described herein, immortalized PMP cell lines that can be stably passaged do not exist due to the indolent biological behavior of PMP cells 126,127 .Consequently, although some of the constituents in the TIME of PMP have been preliminarily identified, the exact composition of the TIME as well as the specific mechanisms of interaction between PMP cells and immunosuppressive factors have not been elaborated, which impedes the establishment of novel immunotherapies for PMP 74,77,79 .
Although PMP is a rare disease worldwide, the origin and progression of PMP have been identified in general terms through the efforts of oncologists.Based on existing research, mucinous tumor cells originating from the appendix or ovary are the key culprits in PMP.PMP cells usually have mutations in the KRAS and GNAS genes, the products of which are involved in the Ras-Erk-Akt and cAMP-PKA signaling pathways, respectively 128 .PMP has unique pathologic characteristics.Specifically, the tumors are prone to widespread peritoneal dissemination but rarely show invasive or infiltrative behavior and PMP is accompanied by extreme mucus secretion.The latter factor is closely related to GNAS mutations, which lead to the accumulation of large amounts of jelly-like mucus ascites in the peritoneal cavity.In advanced stages, mucus sclerosis causes the adhesion and dysfunction of multiple abdominal organs, resulting in decreased quality of life for patients and difficult surgery for surgeons.Therefore, regardless of what treatment method is applied, measures should be taken to target the mucus secretion characteristics of PMP, such as the application of mucolytic strategies [129][130][131] .
Another central issue covered herein is that PMP is characterized by an immunosuppressive microenvironment, which is a common feature of many tumors.The mobilization of the immune system to eliminate PMP might ultimately represent the most efficient strategy.At present, methods for PMP immunotherapy remain limited, although they methods show great developmental potential.A single treatment method may be unsustainable and future breakthroughs might focus on the combined treatment of mucolysis and immunotherapy.

Figure 1
Figure 1 Tumor progression (top) and pathologic pattern (bottom) of pseudomyxoma peritonei (PMP).(A) Greater than 90% of PMPs are derived from the appendix, with the ovary being a secondary source.Other rare sites of origins include the intestinal tract and urachus.(B) In appendix-derived PMP, the perforation and rupture of an appendiceal mucinous tumor lead to release of free tumor cells and gelatinous mucus, which are disseminated throughout the peritoneum.The tumor cells implant on the peritoneal surface and begin to form a PMP focus in a process termed "redistribution".(C) The nascent PMP focus might rupture and repeat the process, resulting in multiple intraperitoneal metastases.(D) The massive mucin produced by PMP cells generates gelatinous ascites in the peritoneal cavity, and the accumulation and sclerosis of the gelatinous ascites lead to abdominal pain, abdominal distension, and intestinal obstruction.The dashed box shows the classification of PMP in order of malignancy, as follows: (E) acellular mucin (a descriptive term), scale bar = 100 μm; (F) low-grade mucinous carcinoma peritonei (LMCP), which is synonymous with disseminated peritoneal adenomucinosis (DPAM), scale bar = 25 μm; (G) high-grade mucinous carcinoma peritonei (HMCP), which is synonymous with peritoneal mucinous carcinomatosis (PMCA), scale bar = 50 μm and (H) high-grade mucinous carcinoma peritonei with signet ring cells (HMCP-S), which is synonymous with peritoneal mucinous carcinomatosis with signet ring cells (PMCA-S), scale bar = 100 μm.The schematic diagram was created with BioRender.com.

TheFigure 2
Figure 2 The GNAS mutation and its secretion-promoting effect on MUC2.(A) The most frequent GNAS mutations detected on chromosome 20q13.32are two single nucleotide variations, which are located at Chr20:57,484,420 and Chr20:57,484,421, corresponding to c.602 G > A and c.601 C > T, respectively.These genomic mutations lead to Cys 201 or His 201 mutations, respectively (B), in the Gsα subunit, which results in changes to the tertiary structure within the GTPase domain (C).(D) The mutant Gsα subunits are unable to hydrolyze GTP, maintaining the protein in a constitutively activated state and continuously activating adenylyl cyclase and downstream cAMP-dependent pathways.(E) The promoting signals activate expression of MUC-related genes, in part through activation of CREB, leading to overproduction of mucins, including MUC2, the most prominent and characteristic mucin of PMP, as well as MUC5B and MUC5AC.(F) The mucin-to-cell volume ratio can increase to 10:1.Mucins are highly glycosylated proteins, increasing the viscoelasticity of mucus.MUC2, for example, undergoes N-and O-glycosylation as a dimer in the rough ER and Golgi apparatus, respectively.The chemical structural formula of glycosylation sites in MUC2 is shown in the figure.(G) The mucin macromolecule would be secreted by exocytosis.GNAS, guanine nucleotide-binding protein alpha subunit; MUC2, mucin 2; MUC5B, mucin 5B; MUC5AC, mucin 5AC; GTP, guanosine triphosphate; CREB, cAMP-response element-binding protein; ER, endoplasmic reticulum.Schematic diagram was created with BioRender.com. Figure 2C is from the RCSB PDB (RCSB.org) of PDB ID 6AU6.

Figure 3
Figure 3 The tumor microenvironment (TME) of PMP.The single isolated and clustered, fibroblast-like, or spherical-shaped cells from an appendiceal or ovarian mucinous tumor are wrapped in mucus, float in ascites, and colonize the peritoneum.Once the tumor cells adhere, the tumor cells begin to reproduce, develop a typical goblet cell-like morphology, and restore the apical-basal polarity and tight cell-to-cell junctions.The tumor cells exhibit an epithelial-mesenchymal plasticity phenotype to better adapt to the peritoneal environment according to the different phases of tumorigenesis.These cells attract immune cells, including monocytes/macrophages, B-and T-lymphocytes, natural killer cells and dendritic cells, and stromal cells, including tumor-associated fibroblasts and mesenchymal stromal cells.The tumor infiltrate has been shown to contain CD3 + T lymphocytes, CD20 + B lymphocytes, and CD68 + macrophages.The assumptive immune landscape of TME inPMP is presented based on existing evidence.Specifically, the localized distribution of some cytokines and chemokines has been found in TME of PMPs, such as IL-6 aggregating in stroma, IL-8 in adipose tissue, and IP-10 and MCP-1 in PMP cells.Moreover, some specific cytokines and chemokines in ascites of PMP patients are 200-fold higher than physiologic levels, including IL-6, IL-8, IP-10, MCP-1, and MIP-1α.The detailed interaction of these immune components in the TME requires further research.PMP, pseudomyxoma peritonei; CD, cluster of differentiation; IL-6, interleukin-6; IL-8, interleukin-8; IP-10, interferon gamma-induced protein 10; MCP-1, monocyte chemotactic protein-1; MIP-1α, macrophage inflammatory protein-1α.Scale bar = 25 μm.The schematic diagram was created with BioRender.com.

Figure 5
Figure 5 Fishbone diagram of immune-associated therapies and research strategies for PMP.