Studies indicate that heterotypic cell-in-cell(hCIC) structures occur in the penetration of immune cells into tumor cells or thymic nurse cells and others, discovered in the intestine epithelia by a German scholar 150 years ago(10). In tumor tissues, tumor cells are an important target of hCIC, and various tumor cells seem to be able to internalize immune cells. For example, HNSCC, melanoma, gastric cancer, ductal carcinoma of salivary gland, breast cancer, liver cancer and other tumor cells(11, 12). Similarly, a variety of immune cells can enter into tumor cells, such as neutrophils(11–14),NK cells, T lymphocytes, LAK cells, B cells et al., among which neutrophils were mostly reported. hCIC structure is closely related to tumor, which is not only common in various tumor tissues, but also significantly correlated with the pathological grade of various tumors, suggesting that the formation of hCIC structure may play an important role in the occurrence and development of tumor. In addition, studies in Jerry's laboratory have shown that thymic nurse cells can internalize the developing T cells to form the hCIC structure to prevent T cells from apoptosis, and this process is also involved in the negative and positive selection of T cells by thymic nurse cells(15), which indicates that hCIC plays an important role in the development and maturation of T cells.
Heterotypic neutrophil-in-tumor structures formed by neutrophils penetrating into tumor cells are often noted in the solid tumors with HNSCC included(16, 17). In our study, we cocultured TSCC cell line-CAL33 with neutrophils in vitro to form neutrophil-in-tumor strutures. We found that well-differentiated tumor cells have higher ability to internalize more neutrophils than poorly-differentiated tumor cells. Though in the association between clinical pathologic characteristics and FNiT we noted that tumor grade was positively correlated with FNiT, and well-differentiated tumor tended to have low FNiT. That is, frequency of neutrophil-in-tumor structures in well-differentiated tumor tissue is lower than that of poorly-differentiated tumor tissue. This is not contradictory to the result of heterotypic cell-in-cell assay in vitro mentioned above. We speculate that the tumor tissues with low FNiT have less neutrophils infiltration than the ones with high FNiT, but the proportion of well-differentiated tumor cells in the former outcompeted that in the latter. This is consistent with the outcome of the patients between with low FNiT and with high FNiT mentioned previously. We could further infer that tumors with more neutrophils infiltration have an adverse prognosis than tumors with less neutrophils infiltration.
The value of FNiT in the prognosis of patients with TSCC has scarcely been investigated to date. A few of papers have reported the existence of neutrophil-in-tumor structures in other tumors of HNSCC. Seza Tetikkurt et al(9) reported a case depicting the presence of the significant neutrophil-in-tumor structures in the squamous cell carcinoma of the hard palate and maxilla which is rarely encountered. Neutrophil-in-tumor structures may predict tumor behavior and the consequences of immune-modulating treatment response in HNSCC. S. C. Sarode et al(16) revealed neutrophil-in-tumor structrues were significantly correlated with poor differentiation of tumor cells and cervical lymph node metastasis. Similar findings of the relationship between FNiT and tumor grade were also noted in current study. Gupta K et al(1) found that cell-in-cell structure was a dependable cytological feature of malignancy. This implied that high FNiT was positively correlated with the malignancy and relevant adverse outcomes of tumors, which was consistent with our views mentioned previously. Furthermore, neutrophil-in-tumor structures have been identified by light microscopy evaluation of pleomorphic cell (giant cell) carcinomas of the intestine, lung, and pancreas, breast carcinomas, invasive micropapillary carcinomas of the ampulopancreaticobiliary region, gastric carcinomas, and lymphomas(11, 14, 18–21).
In our study, we innovatively explored the value of FNiT in the prognosis of patients with TSCC and found that FNiT is significantly associated with DSS. However, the mechanism underlying the association between prognosis as well as clinicopathological characteristics remains unclear. As we proposed above, the FNiT reflects the degree of neutrophil infiltration within the tumor microenvironment. Neutrophils may lead to a significant impact on the tumor microenvironment through cytokines and chemokines that cause inflammatory cell recruitment and activation which could promote tumor cell proliferation, microvascular regeneration and tumor metastasis as a result(7–9). This may better explain the reason why patients with high FNiT tended to have lower 5-year DSS rate than those with low FNiT. The specific role of neutrophils in the pathogenesis of cancer has recently become the subject of research with special focus on the association between tumor microenvironment and tumor progression. Neutrophils may play a crucial role in cancer biology and may act as tumor promotor in tumor progression. Neutrophils may be vital biomarkers for HNSCC and targets to control the progression of HNSCC(8, 22). Mackay HL et al.(23)disclosed that neutrophil-in-tumor structures are positively correlated with poor prognosis in cancer patiets.
In the univariate and multivariate analysis of the predictors for DSS in patients with TSCC, we currently discovered that FNiT, disease stage, lymphovascular invasion and tumor grade were four independent predictors for DSS in the Cox model. However, other candidate predictors such as tumor stage, node stage, metastasis and perineural invasion were not proved to be independently associated with DSS. We did not expect this. Possible speculations may be that the proportions of the positive patient of the node stage, metastasis and perineural invasion parameters were all pretty small, which would have strong error and bias in the survival analysis. Particularly, our data indicated that tumor stage may be correlated with DSS dependent on FNiT. In the research on the association between FNiT and clinicopathological characteristics, we concluded that FNiT was associated with tumor stage(p = 0.027).