Pathophysiology of Mesenteric Fibrosis in Small-intestinal Neuroendocrine NeoplasmsTumor Microenvironment

The potential of tumor invasion and metastasis are mainly attributed to the tumor microenvironment (TME). TME consists of stromal, endothelial, inflammatory cells and extracellular matrix (ECM), in the case of SI-NENs, it is also involved in the development of fibrosis. Desmoplastic reaction is considered as a negative prognostic factor in malignancy. The interplay between tumor cells and surrounding TME is considered responsible for the TME remodeling, which, in turn, enables tumor progression. The local microenvironment in SI-NEN is particularly characterized by a pronounced desmoplastic reaction with limited leucocyte infiltration. Several paracrine amines, peptides and growth factors are involved in this complex process enabling communication of tumor cells with the surrounding stromal cells and promoting the synthesis of profibrotic factors [1, 9].

One of the main factors considered as the driver for the development of MF in SI-NEN is the bioamine serotonin (or 5-hydroxytryptamine, 5-HT). Systemic serotonin secretion by SI-NEN is quantified by the measurement of its metabolite 5HIAA in the urine. However, as local tissue serotonin levels do not always correlate with urinary 5HIAA levels, a paracrine role of this molecule in the development of MF has been suggested. The serotonin role on induction of cardiac fibrosis was confirmed in an in vivo study by Gustafsson et al., in rats that developed heart valve disease after long-term serotonin administration, but for MF no such in vivo experiments are available [10]. In an in vitro study by Svejda et al., serotonin stimulated the fibroblast-like HEK293 cell line proliferation, and synthesis of transforming growth factor β (TGFβ), connective tissue growth factor (CTGF) and fibroblast growth factor (FGF2), whereas administration of a serotonin receptor (5-HT2B) antagonist decreased serotonin and profibrotic growth factor synthesis and secretion [11]. In a study by Blazevic et al., a decreased expression of serotonin-metabolizing enzymes in the stroma of fibrotic mesenteric metastases has been documented, with lower levels of monoamine oxidase A (MAO-A), a key enzyme of serotonin catabolism, in the tumor cells of SI-NEN patients with MF [12].

Fibroblasts

Fibroblasts are one of the major cell types surrounding a tumor mass, and in their activated form, also known as cancer-associated fibroblasts (CAF) play an important role in tumor progression. CAFs are characterized by expression of the α-smooth muscle actin (αSMA) and SI-NENs display in fact a high expression of αSMA, suggesting an important role in SI-NEN growth [13]. CAFs in SI-NEN induce the desmoplastic reaction through autocrine/paracrine action of several growth factors [14]. Chaudhry et al., had documented the expression of TGFβ, FGF2, and platelet derived growth factor (PDGF) in NEN of the digestive tract assuming both a stimulation of the tumor and the stromal cell growth and the respective development of a desmoplastic reaction [14, 15]. In vitro experiments have shown that fibroblasts stimulated from the neuroendocrine cell line BON1 secrete TGFβ, whereas further experiments with the co-culture of the SI-NEN cell line KRJ1 and the fibroblast-like cell line HEK293 promotes the release of several growth factors, such as CTGF, FGF2 and TGFβ [11]. TGFβ secreted by SI-NEN induces CTGF expression from both tumoral cells and stromal fibroblasts and both growth factors stimulate collagen synthesis in activated myofibroblasts (now CAF, also known as stellate cells), expressing αSMA [16]. Interleukin-6 (IL-6) and chemo-attractant protein 1 (MCP-1) are secreted by CAF, inducing tumor proliferation [17]. CTGF promotes fibroblast proliferation, migration, adhesion and ECM formation and is highly expressed in tumoral cells adjacent to the stroma [13, 18]. Bone morphogenetic protein 4 (BMP4), a member of the TGFβ superfamily was further found highly expressed in SI-NEN with pronounced fibrosis, rendering this molecule also suspect for the pathogenesis of MF [19]. Finally, fibroblasts nerve growth factors (NGF), regulating angiogenesis [19].

Endothelial Cells

NENs are highly vascularized tumors, reminiscent of the physiological vascular supply of endocrine tissues, and present the paradox, that well-differentiated neoplasms display higher vascular density than poorly differentiated tumors [20]. The vascular endothelial growth factor (VEGF) system, consisting of VEGF and its receptors VEGFR1 and VEGFR2, is highly expressed in these endocrine neoplasms exerting a relevant paracrine action promoting neovascularization. An aberrant activation of the mammalian target of rapamycin (mTOR) signaling pathway is also acknowledged as a factor promoting angiogenesis in these neoplasms [17]. Interestingly, VEGF inhibition has a low effect on limiting tumor growth implying a resistance of these neoplasms. Besides an intrinsic resistance of these neoplasms, as observed by treatment with the VEGF inhibitor sunitinib, an acquired resistance to antiangiogenic treatment can also appear after an initial effective inhibition of angiogenesis. In this case, the resulting hypoxia stimulates the release of hypoxia-inducible factor 1α (HIF1A), which, in turn, induces alternative proangiogenic factors [18]. Further proangiogenic factors include FGF2 and PDGF. FGF2 stimulates endothelial cell proliferation, but its role on MF remains uncertain, as FGF2 expression was comparable in patients with SI-NEN in comparison to controls but also independent from the extent of MF [19]. PDGF, secreted from endothelial cells, also promotes vascular formation and accentuates NEN proliferation. Interestingly, and similar to the CTGF expression, also PDGF expression is higher in tumor cells adjacent to the stroma [21]. Furthermore, PDGF receptor β expression was found pronounced in stromal cells of metastases [14, 22].

Immune Cells

Immune cells are another major component of the TME, with B- and T-lymphocytes, natural killer cells (NK), macrophages, infiltrating the tumor. Interestingly, in SI-NEN the leucocyte infiltration is significantly lower than in other malignancies [17], with T-regulatory lymphocytes, infiltrating in particular metastases, possibly inhibiting the anti-tumor effect of other T-lymphocytes [23]. NK cells are known to inhibit TGFβ profibrotic activity. Substance P (SP), a tachykinin secreted by SI-NEN antagonizes the anti-fibrotic action of NK cells, possibly contributing to MF [24]. Furthermore, NEN tumors display a disturbed NK cell function with deficient interferon (IFN)-α response [17]. Tumor-associated macrophages (TAM) support tumor development, by suppression of the adaptive immune system and stimulate the secretion of profibrotic factors such as TGFβ, promoting fibrosis. In SI-NEN TAMs strongly express TGFβ and PDGF [22]. The expression of the immune checkpoint molecule programmed death-ligand 1 (PDL1) in SI-NEN is rather low. In line with this observation, with the application of two different monoclonal antibodies, we have recently shown no positive PDL1 expression in SI-NEN [25]. However, PD1 antibodies are currently under investigation for their effectiveness in NEN progression [26]. Unlike this, in a study by Rodrigez et al. a significant IgG4 expression in plasma cells of mesenteric tumor deposits in SI-NEN patients was found suggesting a possible interrelation of MF with the IgG4-related disease [27].

Extracellular Matrix (ECM)

ECM consists of polypeptides and polysaccharides necessary for structural integrity and tissue homeostasis. ECM remodeling plays an important role in tumor development, invasion and finally metastasis. Heparan and chondroitin proteoglycans bind several growth factors and chemokines and their differential regulation during disease progression in SI-NEN might affect the development of fibrosis [28]. Matrix metalloproteinases (MMP) are normally involved in the degradation and remodeling of matrix and promote angiogenesis, and their absence correlated with an aggressive phenotype in an in vivo model of pancreatic NEN (PanNEN), but no data are available on their role in SI-NEN [18]. Collagen III, a further component of ECM, accumulates in proximity to CAFs, as product of these fibroblasts, also contributing to fibrosis [16]. Components of the extracellular matrix further include profibrotic growth factors, such as serotonin, CTGF, TGFβ, PDGF, FGF2, with their role in MF pathogenesis already discussed. Further growth factors, such as insulin like growth factor (IGF-1), epidermal growth factor (EGF) and TGFα might also present an autocrine action relevant for tumor proliferation, as their receptors are present in NEN. Still, their role in MF remains unclear [18]. Recently, sexual dimorphism was observed in the development of MF, with premenopausal women presenting reduced MF and better prognosis, and this fact correlating with an increased estrogen receptor (ER) α and AR expression in the SI-NEN microenvironment [29] (Fig. 2).

Fig. 2

Components affecting the tumor microenvironment and contributing to mesenteric fibrosis in SI-NEN. 5HT2B: serotonin receptor 2B, αSMA: α-smooth muscle actin, CAF: cancer-associated fibroblasts, CTGF: connective tissue growth factor, EGF: epidermal growth factor, EGFR: epidermal growth factor receptor, FGF2: fibroblast growth factor, FGFR: fibroblast growth factor receptor, IFNα: interferon-α, IGF1: insulin-like growth factor 1, IGF1R: insulin-like growth factor 1 receptor, LC: lymphocytes, MMPs: matrix metalloproteinases, NK: natural killer cells, PDGF: platelet-derived growth factor, PDGFR: platelet-derived growth factor receptor, SP: substance P, TAM: tumor associated macrophages, TGFβ: transforming growth factor β, TGFβR: transforming growth factor β receptor, VEGF: vascular endothelial growth factor, VEGFR: vascular endothelial growth factor receptor

Signaling Pathways

Well-known pathways (Hedgehog, Notch, Wnt/β-catenin, integrins), implicated both in fibrotic diseases and cancer have also been studied in SI-NEN. In an immunohistochemical study (IHC) of SI-NEN, the expression of Sonic Hedgehog ligand was found positive in almost three quarters of the patients [30]. In a further study focusing on the Notch pathway, no significant difference in the transcription of NOTCH1, NOTCH2, NOTCH3, HES1, HEY1 and HEY2 was documented in a series of 8 SI-NEN, but upregulated ASCL1 transcription was found in half of the tumors [31]. However, IHC staining of NOTCH1 and HES1 on a tissue micro-array of 31 SI-NEN was negative [32]. In the case of the wnt/β-catenin signaling pathway, no correlation of the severity of MF with the β-catenin expression was found [19], whereas a further study showed only normal membranous expression of β-catenin in SI-NEN [30]. In an in vitro co-culture model of SI-NEN cells KRJ-I with HEK293 conditioned media the integrin subunit alpha v (ITGAV) was significantly upregulated, whereas in HEK293 cells with KRJ-1 conditioned media Metalloproteinase 8 (MMP8), TGFβ3 and the integrin ITGB8, all with a role on fibrosis development, were significantly downregulated. The activation of the integrin pathway was further validated in a cohort of SI-NEN, where a significant upregulation of the integrin pathway genes TGFβ1, collagens COL1A1, COL3A1, fibronectin 1, and integrins ITGAV, and ITGAX was documented [33].

Fibrosome and Other Profibrotic Molecules

Laskaratos et al., recently introduced the definition of fibrosome, including a subset of circulating transcripts from the gene molecular signature of the NETest with known roles in fibrosis, namely: CTGF, the complement regulator CD59, amyloid precursor-like protein 2 (APLP2), frizzled homologue 7 (FZD7) and BNIP3L and documented that the presence of APLP2, BNIP3L, CD59 and CTGF transcripts could independently predict the presence of MF in SI-NEN at a statistically significant level [34]. However, in a further IHC study, no correlation of APLP2, BNIP3L, or CD59 tissue expression with the presence of MF could be demonstrated [35]. In a proteomic analysis comparing samples from SI-NEN patients with and without MF, differential protein expression was only documented in the mesenteric stroma, where Collagen α1(XII) (COL12A1) and complement C9 expression had higher abundance in MF samples [36]. Finally, in a biomarker panel study by Graf et al., differential gene expression between SI-NEN patients with or without MF included increased COMP and COL11A1 gene expression in the stroma of MF patients, and decreased HMGA2, COL6A6, and SLC22A3 expression in lymph node metastases of MF patients, with COMP being strongly associated with the known profibrotic factor TGFβ [37].

Clinical Presentation and Diagnosis of Mesenteric Fibrosis in Small-intestinal Neuroendocrine Neoplasms

Due to their indolent nature, SI-NEN are often diagnosed at advanced stages, and their clinical manifestations frequently occur due to the extensive MF adjacent to local lymph node metastases and locoregional mesenteric tumor deposits. Extensive MF in SI-NEN patients can cause acute symptoms due to mesenteric vessel obstruction leading to intestinal ischemia, and intestinal obstruction. Abdominal pain, worsening of diarrhea and ascites are the main acute symptoms of MF. Postprandial abdominal pain and diarrhea can further induce malabsorption and lead to nutritional deficiencies, in particular of fat-soluble vitamins, dehydration and electrolyte imbalances [9]. Furthermore, MF can extend to the retroperitoneum. Here, it is hypothesized that biogenic amines escaping liver inactivation are the main triggers for the development of retroperitoneal fibrosis, leading to obstructive uropathy, hydronephrosis up to renal failure at later disease stages [38].

The radiological findings in the case of MF in SI-NEN are rather peculiar. Pantongrag et al., described a classic trias of signs characteristic of SI-NEN including a calcified mesenteric mass, radiating strands, and adjacent bowel-wall thickening. A mesenteric mass with radiating strands of soft-tissue on CT imaging is now considered a pathognomonic feature of SI-NEN related MF [39]. Further radiological manifestations suggestive of MF in a cohort of SI-NEN patients include mesenteric vessel encasement, presence of hepatic metastases and large hepatic tumor burden, mesenteric mass with coarse or fine calcification, “indrawing” of tissues and “misty” mesentery [40, 41]. The presence and extent of lymph node metastases and of MF in SI-NEN patients play an important role for surgical planning, so that radiological assessment should be optimized. Therefore, functional imaging with 68Ga-DOTATOC-PET/CT can improve the detection of lymph node metastases in MF. While 18F-FDG-PET/CT is only helpful in the visualization of aggressive NEN, in a case report, 18F-FDG uptake was documented in a retroperitoneal fibrotic mass of a patient with a carcinoid tumor [42].

The histological assessment of MF is based on the section of the surgical specimen with the highest amount of fibrous tissue. Histologically, SI-NEN fibrosis presents hypocellular, with prominent hyalinization and development of foci of calcification. Histological distinction between positive lymph nodes and MTD can be difficult in many cases and irregular shape and nerve/vessel entrapment can serve as a marker of MTD [43].

Surgical Management of Mesenteric Fibrosis

The fibrotic process, driven by tumor-associated desmoplastic reactions, leads to mesenteric retraction and vascular compromise, often necessitating surgical intervention. Surgery is often required to manage symptoms and prevent complications arising from MF. Palliative surgery is indicated for patients experiencing intestinal obstruction, ischemic complications, and severe abdominal pain [44]. Similarly, it has been shown that 82% of patients undergoing laparotomy for midgut carcinoid tumors experience symptom relief, supporting a proactive surgical approach [45]. Despite this, no significant survival benefit has been observed with surgical resection compared to non-surgical management, emphasizing its primarily palliative role. Surgery should aim at a systematic lymphadenectomy avoiding a short bowel syndrome.

Surgical Techniques

All localized SI-NENs should undergo surgery with proper lymph node dissection. The definition of surgical resectability should take into account the degree of arterial involvement in lymph-node metastases. According to the classification of Ohrvall and colleagues Stage I consists of tumors located close to the intestine, stage II includes tumors involving arterial branches close to their origin in the mesenteric artery, stage III comprises tumors extending along, without encircling, the superior mesenteric artery trunk, and finally stage IV refers to tumors that extend retroperitoneally, behind or above the pancreas, or grow around the mesenteric artery and involve the origin of proximal jejunal arteries on the left side of the superior mesenteric artery [46].

A combination of primary tumor and mesenteric mass resection is recommended for curative intent and to prevent debilitating MF [47]. The preferred surgical approach aims at a balance between oncologic radicality and preservation of bowel function. A mesenteric-sparing technique is often favored to avoid excessive bowel resection.

Vessel-sparing lymphadenectomy (VS-LA) seems to be preferred over conventional lymphadenectomy (Con-LA) due to its superior postoperative outcomes. VS-LA has shown to result in shorter bowel resections (median 40 cm vs. 65 cm, p = 0.007), lower rates of postoperative complications (4% vs. 28%, p = 0.02), and comparable oncological outcomes (R0 resection rate: 72% vs. 84%). For this reason, the surgical approach should not be a “pizza pie” approach (including a large intestinal resection), but a retrograde VS-LA [48]. In a recent retrospective study, it has been shown that the resected bowel specimen is shortened by about half, when using this technique [49]. In cases where complete resection is unfeasible due to extensive vascular involvement, intestinal bypass procedures may be performed to alleviate obstruction. However, this approach is reserved for patients with severe symptoms, as it does not provide a survival advantage.

Staging and Surgical Feasibility

In the context of MF resectability, mesenteric disease can be categorized into three subtypes: type A including the resectable mesenteric disease not involving the mesenteric root; type B including a borderline resectable disease with fibrosis near but not encasing major mesenteric vessels; type C including locally advanced, unresectable disease with complete encasement of the superior mesenteric artery (SMA) and vein (SMV). For Types A and B, surgery is generally feasible with experienced surgical teams. Type C lesions, however, may necessitate alternative palliative strategies, such as somatostatin analogs, nutritional support, and palliative bypass [50]. A suggested algorithm for surgical intervention can be found in Fig. 3.

Fig. 3

Suggested algorithm for surgical intervention in mesenteric fibrosis in SI-NEN. 5HIAA: 5-hydroxyindoleacetic acid, CgA: Chromogranin A, LN: lymph nodes, PRRT: peptide receptor radionuclide therapy, SSA: somatostatin analogues

Surgical Outcomes

Several studies have assessed the effectiveness of surgical intervention for MF. It is shown that 41.4% of patients exhibited MF, with older age, high 5-HIAA levels, and large mesenteric masses being independent predictors. The authors reported no significant survival benefit from metastasectomy of mesenteric masses or prophylactic surgery, suggesting that surgical intervention should be symptom-driven rather than routinely performed [44]. In contrast, it is demonstrated that mesenteric tumor dissection with vessel preservation significantly improved symptoms and quality of life, even in cases initially deemed inoperable [49]. Similarly, lymphadenectomy should be carefully tailored to balance tumor clearance with bowel preservation, as aggressive resection can lead to complications such as short bowel syndrome [47].

Long-term Outcomes and Survival

Median survival after laparotomy was 9 years, with a subset of patients achieving 12-year survival. Notably, survival was independent of surgical intervention in patients with extensive metastases, emphasizing the palliative nature of surgery [45]. In the context of quality of life and functional outcomes, patients who underwent VS-LA had significantly lower postoperative complication rates (4% vs. 28%) and lower incidence of postoperative diarrhea (4% vs. 40%) [48]. This highlights the importance of preserving intestinal length and vascular integrity during surgery. SI-NEN should be operated in high-volume centers of excellence since it was shown that the 90-day mortality after surgery is higher in low-volume centers compared to high-volume hospitals (4% vs. 1%) [51].

Medical Management of Mesenteric FibrosisSomatostatin analogues

Somatostatin analogues (SSAs) are considered a first-line therapy for functioning NEN [52, 53]. Nearly 80% of carcinoid tumors express somatostatin receptors (SSTRs). SSAs can attenuate the symptoms of carcinoid syndrome including diarrhea and flushing via their anti-secretory effects [