Ultrastructural Features of Amoeboid Tumor Cell–Unmyelinated Nerve Fiber Interactions in Early Gastric Cancer: A Case Report Within the Context of Cancer Neuroscience

Abstract

Background: Perineural invasion (PNI) is a recognized pathway for cancer spread and is associated with poor outcomes in gastric cancer. However, the initial morphological characteristics of tumor–nerve interactions in early gastric cancer, particularly at the ultrastructural level, remain insufficiently defined. Case Presentation: We report a case of a 49-year-old man diagnosed with type IIc early gastric cancer. Histological examination revealed a combined poorly cohesive carcinoma (PCC)-NOS/signet-ring cell (SRC) histotype. Tumor invasion reached the middle third of the submucosa and was accompanied by a mature desmoplastic reaction, with metastases identified in two perigastric lymph nodes (pT1bN1M0). Transmission electron microscopy (TEM) revealed unmyelinated nerve fibers embedded within the submucosal desmoplastic stroma, in close proximity to infiltrating neoplastic cells. Several tumor cells exhibited cytoplasmic projections ranging from single extensions to multiple prominent pseudopods, resulting in an amoeboid morphology. Notably, an unmyelinated nerve process was observed within a cytoplasmic invagination of an individual tumor cell. Conclusions: Taken together, these ultrastructural findings provide novel and previously undescribed morphological evidence of a specific interaction between amoeboid tumor cells and peripheral unmyelinated nerve fibers within the submucosal desmoplastic stroma of early gastric cancer. The biological and clinical significance of this interaction in the early stages of perineural invasion warrants further investigation.

1. Background

The tumor microenvironment is a dynamic and heterogeneous ecosystem consisting of immune cells, neurons, lymphatic and blood vessels, and the extracellular matrix that surrounds the tumor. Within this intricate network, tumor cells communicate with host cells through direct intercellular contacts or paracrine signaling within specialized regions known as tumor niches, which promote cancer progression and metastasis [1,2,3].

The innervated niche is one of these compartments that has recently drawn particular attention. It refers to a microenvironment rich in sympathetic, parasympathetic, or sensory fibers, often characterized by small diameter axons and dense neurochemical signaling that continuously interact with tumor and stromal cells via neurotransmitters and neuropeptides released into the extracellular space [4]. Evidence from both human and experimental studies indicates that the peripheral nervous system may play a crucial role in various malignancies, influencing their initiation and progression [5,6,7,8,9,10]. These discoveries have led to the development of cancer neuroscience, an interdisciplinary field investigating how neural signaling affects tumor dynamics and how cancer, in turn, modulates neural circuits within the tumor microenvironment [11,12,13].

Perineural invasion (PNI), classically described as the invasion of cancer cells in, around, and along nerves, is a distinct pathway of tumor dissemination and an adverse prognostic indicator in several cancers [14]. Standard hematoxylin–eosin-stained sections, frequently complemented by histochemical and immunohistochemical methods, enable surgical pathologists to detect PNI, typically assessed at the level of Auerbach’s (myenteric) plexus [15] or major nerve trunks situated within the subserosal soft tissue [16].

Toluidine blue-stained semithin resin sections offer a more detailed evaluation of neural morphology than routine paraffin sections, whereas transmission electron microscopy (TEM) is the gold standard for identifying unmyelinated fibers, which are not reliably recognized by light microscopy because of their small caliber (approximately 0.1–3 μm diameter) [17,18]. TEM also facilitates the examination of neuroeffector junctions, which are specialized regions where autonomic postganglionic axons establish dynamic connections with diverse non-neuronal effector cells, including smooth muscle, glandular, and immune cells [19]. In these junctions, neurotransmitter release typically occurs from swellings (varicosities) along autonomic nerve fibers, rather than exclusively at the terminal end, and diffuses as paracrine signaling over short distances to reach target cells [19,20].

The neural component of the tumor microenvironment has been the subject of relatively limited ultrastructural investigations, the majority of which have focused on neural structures in proximity to tumors. Zauszkiewicz-Pawlak et al. [21] analyzed the ultrastructure of the myenteric plexus in colonic areas distant from colorectal carcinomas, comparing them with plexuses situated near the tumor tissue. Other researchers have documented ultrastructural characteristics of PNI in peritumoral areas, typically involving larger nerve trunks or plexuses in more advanced stages [22,23,24]. However, to the best of our knowledge, no ultrastructural studies specifically examining nerve–tumor interactions in human gastric carcinoma have been documented yet.

The present report details an early gastric cancer in which TEM demonstrates unmyelinated nerve fibers embedded within the submucosal desmoplastic tumor stroma, establishing highly intimate associations with amoeboid tumor cells. These observations are analyzed within the wider context of cancer neuroscience [11] and are presented as hypothesis-generating ultrastructural evidence of specific tumor–nerve interactions within the submucosal compartment of early gastric cancer.

2. Case Report

Between 1998 and 2005, gastric carcinoma specimens examined at the Department of Human Pathology of the University Hospital “G. Martino” in Messina were routinely processed for both light microscopy and TEM. This integrated diagnostic protocol was predominantly utilized for specific neoplasms in which ultrastructural analysis could aid in differential diagnosis (e.g., neuroendocrine or hepatoid carcinomas, non-epithelial tumors).

Light microscopy and TEM were performed on paired tissue samples obtained contemporaneously from fresh surgical specimens and immediately fixed in parallel, thereby avoiding fixation delays or the use of archival material for ultrastructural evaluation. For each case, fresh tumor tissue was sectioned into two contiguous portions using a razor blade, allowing the acquisition of matched samples for conventional histology and electron microscopy from the same anatomical region of the lesion. One portion was fixed in 10% neutral-buffered formalin and embedded in paraffin, in addition to supplementary tissue samples obtained from the tumor, perigastric lymph nodes, and the surgical resection margins. Hematoxylin and eosin staining was employed for standard histological evaluation. The contiguous second portion was cut into small fragments (1–2 mm in diameter) and processed for TEM. These samples were fixed in 3% phosphate-buffered glutaraldehyde (pH 7.4), subsequently post-fixed in 1% osmium tetroxide, dehydrated using a graded ethanol series, and embedded in Araldite. The patient described in this report was part of this diagnostic series.

A 49-year-old male was admitted to the University Hospital “G. Martino” in Messina, Italy, on 15 February 2004, with a two-month history of epigastric pain. Upper endoscopy revealed an approximately 2.0 × 1.5 cm type IIc depressed lesion situated at the incisura angularis. Targeted biopsies revealed the presence of signet-ring cell (SRC) carcinoma, while supplementary biopsy samples obtained from the lesser curvature of the antrum and body were positive for Helicobacter pylori using Giemsa staining. Preoperative staging, comprising chest radiography, abdominal ultrasonography, and contrast-enhanced computed tomography, indicated no signs of distant metastases or locoregional invasion. A subtotal gastrectomy and regional lymphadenectomy were later performed on the patient. The postoperative recovery proceeded without complications. Adjuvant therapy was not implemented, according to therapeutic protocols of 2004. During the long-term follow-up, the patient remained asymptomatic, with no clinical or radiological indications of recurrence or metastatic disease for a 15-year duration.

The resected stomach showed a depressed lesion measuring 2.0 × 1.5 cm at the incisura angularis. The tumor infiltrated the middle third of the submucosal layer (Figure 1A), and all resection margins were devoid of neoplastic invasion. Two of the eighteen perigastric lymph nodes revealed metastases, but there was no light microscopic evidence of venous or PNI. The neoplasm was reclassified as pT1bN1M0, stage IB (early gastric cancer), in accordance with the American Joint Committee on Cancer TNM staging system for gastric cancer (Eighth Edition) [25]. The tumor consisted of individual cells or small clusters lacking glandular differentiation, a characteristic corresponding with a poorly cohesive carcinoma (PCC)-NOS, as defined by the WHO classification [26]. It displayed different cytological types, including SRC and histiocytoid components. Based on the presence of an SRC component constituting more than 10% but less than 90%, PCC-NOS was subsequently subclassified into combined PCC-NOS/SRC in accordance with the recent European consensus established by the International Gastric Carcinoma Association [27]. SRC was the most prevalent type of cell that infiltrated the mucosal layer (Figure 1B). On the other hand, histiocytoid tumor cells, sparsely distributed in a desmoplastic stroma, were mainly detected in the submucosal layer (Figure 2A). Focal eosinophil infiltration was identified in these regions. Based on the absence of myxoid stroma or keloid-like collagen bundles (Figure 1A), the desmoplastic reaction was designated as “mature” in accordance with Ueno’s histopathological classification [28]. In the paired specimens embedded in Araldite, submucosal infiltration by histiocytoid tumor cells accompanied by a mature desmoplastic response was confirmed in semithin sections stained with toluidine blue (Figure 2B and Figure 3). There was marked variability in the density of submucosal tumor cells. In some areas, only scattered histiocytoid elements were present within the desmoplastic stroma (Figure 2B), whereas in other fields, the stroma was densely populated by crowded but still poorly cohesive histiocytoid tumor cells (Figure 3). These malignant cells exhibited a single, enlarged, round-to-irregular nucleus with a prominent nucleolus (Figure 2B and Figure 3).

We adopted a strict correlative sampling procedure to ensure that the tissue examined under TEM corresponded to the tumor component infiltrating the submucosal layer. This approach was based on a stepwise comparison between hematoxylin–eosin-stained sections, semithin toluidine blue-stained sections, and TEM. Submucosal tumor invasion was identified on hematoxylin–eosin-stained sections (Figure 1A,B and Figure 2A) and precisely localized for ultrastructural analysis using corresponding semithin toluidine blue-stained sections (Figure 2B and Figure 3). Araldite blocks containing these predefined regions were then sectioned for TEM. Using the reference photomicrographs obtained from the semithin sections, the same anatomical structures were subsequently identified, examined, and documented at the ultrastructural level, using a JEOL 1200 TEM (JEOL, Tokyo, Japan). Within the analyzed specimen, the muscularis propria was consistently devoid of neoplastic infiltration in semithin sections, thereby corroborating the diagnosis of an early-stage gastric carcinoma and confirming that the ultrastructural findings were obtained within the context of submucosal invasion.

TEM identified unmyelinated nerve fibers embedded within the submucosal desmoplastic stroma, in close proximity to infiltrating neoplastic cells. A morphological continuum of nerve–tumor interfaces was noted, varying from mere proximity to highly intimate connections characterized by considerable narrowing of the intercellular gap (Figure 4). Moreover, tumor cells often displayed cytoplasmic protrusions oriented toward adjacent unmyelinated fibers. Examples of these cytoplasmic protrusions from tumor cells are illustrated in Figure 4, which shows a finger-like projection from the cytoplasm of a tumor cell, and in Figure 5, where tumor cells extended two broad cytoplasmic pseudopods toward nearby unmyelinated nerve fibers, while the axonal profiles did not show varicosities or clustered synaptic vesicles. These cytoplasmic pseudopods conferred an amoeboid shape in some tumor cells. Specifically, Figure 6 illustrates an amoeboid tumor cell, situated near two distinct unmyelinated nerve fibers, arranged in a bipolar configuration.

At greater magnification, no distinguishable intercellular fissure could be observed between the unmyelinated nerve fiber and the tumor cell. Moreover, a neural process appeared to be accommodated within a cytoplasmic invagination of the tumor cell (Figure 7), resulting in highly intimate nerve–tumor interactions within the submucosal desmoplastic region. Furthermore, in the examined sections, the axonal profile closely apposed to the tumor cell did not show focal swelling or a clustered accumulation of synaptic vesicles and is therefore best interpreted as a non-varicose segment of an unmyelinated axon.

Figure 8 provides a schematic summary of the anatomical context and the tumor–nerve interaction patterns documented in this case to facilitate the integration of the histological and ultrastructural observations described above.

3. Discussion

We report a case of type IIc early gastric cancer with submucosal invasion, classified as a combined PCC-NOS/SRC carcinoma, with an SRC component exceeding 10% but less than 90% [26,27].

This case exhibits clinicopathological characteristics, including poorly cohesive histology with SRC and histiocytoid cells, significant mature desmoplasia in the submucosa, and an excellent long-term prognosis, similar to the low-grade diffuse gastric cancer subtype described by Chiaravalli et al. [29], which is correlated with a relatively favorable outcome. It also has histological similarities to the PCC-NOS/SRC carcinomas described by Roviello et al. [30]. In this study, patients with PCC-NOS/SRC exhibited superior survival compared to those with PCC-NOS.

We examine our case of early gastric cancer within the context of the emerging discipline of cancer neuroscience, which integrates the study of the nervous system with tumor biology [11,12,13]. The peripheral nervous system of the stomach predominantly comprises a well-developed intermuscular ganglionated plexus (Auerbach’s plexus) that provides innervation to the gastric mucosa, submucosa, and smooth muscle tissue [31]. In contrast to the small and large intestines, the human stomach lacks a prominent submucosal plexus (Meissner’s plexus), possessing only a limited number of ganglia and a few unmyelinated nerve fibers [31,32]. PNI is more commonly detected in advanced gastric cancer within the myenteric plexus or extramural nerve trunks, whereas it has been rarely reported in early gastric cancer [33]. However, to the best of our knowledge, no ultrastructural studies regarding nerve–tumor interactions in early gastric cancer have yet been documented.

Cancer cell invasion is characterized by two main types of individual tumor cell migration: mesenchymal (fibroblast-like) and amoeboid [34]. In a recent study, Marcadis et al. [35], utilizing a murine model of PNI within the sciatic nerve, demonstrated that tumor cells rely exclusively on an amoeboid mode of migration to achieve rapid PNI. Furthermore, these authors emphasize that therapeutic interventions can specifically target this mode of migration [35]. In our observations, some tumor cells in contact with nerve fibers displayed a variety of cytoplasmic protrusions, ranging from a single protrusion to multiple large pseudopods, which give rise to an amoeboid morphology. It is important to note that the plasma membranes of the amoeboid tumor cell and of an adjacent unmyelinated nerve fiber are in direct contact, with no visible intercellular gap. Additionally, a nerve process appears to be located within a cytoplasmic invagination of the tumor cell. In the examined sections, this axonal profile did not exhibit focal swelling or a clustered accumulation of synaptic vesicles and is therefore best interpreted as a non-varicose segment of an unmyelinated axon. Taken together, these features do not support the interpretation of this interface as a synapse-like junction between nerve and tumor cell. Given that axonal varicosities are dynamic and distributed segmentally, varicosity-mediated signaling along the same fiber cannot be conclusively ruled out; our negative findings pertain solely to the regions included within the examined sections for TEM. On the other hand, the amoeboid configuration of the neoplastic cells resembles that reported in experimental models of PNI [35]. In this context, the absence of disease recurrence despite long-term follow-up suggests that early and localized tumor–nerve interactions, as observed in this case, do not necessarily correspond to an aggressive or progressive perineural invasive process in early-stage gastric cancer. Accordingly, our findings provide novel ultrastructural evidence of a specific pattern of interaction between amoeboid tumor cells and unmyelinated nerve fibers within the submucosal tumor compartment. Further studies will be required to determine whether this ultrastructural configuration represents an initial submucosal stage of PNI.

Limitations

The diagnosis of early gastric cancer was determined using histopathological criteria, based on the localization of the neoplastic proliferation to the mucosa and superficial submucosa, demonstrated by the low-magnification histological sections provided (Figure 1A,B). Endoscopic or gross macroscopic photographs of the resected specimen were unavailable for this retrospective case; however, the pathological definition of early gastric cancer is based on the depth of tumor invasion detected by light microscopy rather than macroscopic appearance.

In conclusion, we report a rare case of early gastric cancer in which TEM revealed early interactions between amoeboid tumor cells and unmyelinated nerve fibers within the submucosal layer. These observations extend current knowledge on tumor–nerve relationships in early gastric cancer and provide hypothesis-generating ultrastructural evidence that nerve-associated amoeboid tumor cell invasion may represent one of the possible modes of spread within the gastric tumor microenvironment. Further studies are needed to determine whether similar submucosal patterns of tumor–nerve interaction can be documented in additional cases of PCC-NOS advanced gastric carcinoma, particularly in tumors with desmoplasia and PNI along the myenteric (Auerbach’s) plexus.