Cell-in-cell structures are more potent predictors of outcome than senescence or apoptosis in head and neck squamous cell carcinomas

Hannah Schenker, Maike Büttner-Herold, Rainer Fietkau, Luitpold V Distel, Hannah Schenker, Maike Büttner-Herold, Rainer Fietkau, Luitpold V Distel

Abstract

Background: This study sheds light on cell inactivating processes with focus on the phenomenon of cell-in-cell (CIC). Cell-in-cell describes a cell process where one cell is being engulfed by another non-professional phagocyte. We determined frequency and prognostic impact of CIC structures (CICs) as well as of senescent and apoptotic cells in head and neck squamous cell carcinomas (HNSCC).

Methods: These different forms of cell inactivation as well as the proportion of proliferating and tumor cells were assessed in 169 pre-radiochemotherapy biopsies and 32 post-therapy tumor resections by immunohistochemistry of tissue microarrays. Four consecutive cancer sections were stained with antibodies specific for E-cadherin for CIC detection, cleaved caspase-3 for apoptosis, H3K9Me for senescence and Ki67 as a proliferation marker. Positive events were quantified in corresponding tumor areas.

Results: CICs were found in 55.5%, senescent cells in 67.1% and apoptotic cells in 93.3% of samples. While no prognostic impact of apoptotic and senescent cells was observed, CICs turned out to significantly influence overall-survival (p = 0.016) with a lack of CICs being prognostically beneficial. There was no correlation between CICs and apoptosis and 98.9% of CICs were negative for cleaved caspase-3.

Conclusion: CIC formation is a frequent event in HNSCC and a superior predictive marker compared to senescence and apoptosis. Independence of CIC and apoptosis and the adverse prognosis associated with numerous CICs lead to the assumption that CICs might take up necrotic rather than apoptotic cells preventing an adequate antitumoral immune response that would otherwise be initiated by necrotic cells through damage-associated molecular pattern molecules.

Keywords: Apoptosis; Cell death; Cell-in-cell; HNSCC; Proliferation; Senescence.

Figures

Fig. 1
Fig. 1
Frequency of CIC structures, senescent and apoptotic cells assessed by immunohistochemistry. a E-Cadherin staining labelling numerous CIC structures in HNSCC and magnifications of indicated region. (ai) Schematic drawing of a CIC structure illustrating defining criteria: complete encirclement of the inner cell by the host cell membrane, circular shape of the inner cell and semilunar host cell nucleus. b Cleaved caspase-3 labeled HNSCC and magnifications of indicated region showing CIC structures negative for cleaved caspase-3. c H3K9ME labeled HNSCC showing senescent cells and magnifications of indicated region. d Ki67 labeled HNSCC showing proliferating cells and magnifications of indicated region. Comparative analysis of (e) CIC structures, (f) senescent cells, (g) apoptotic cells, (h) proliferating cells and (j) tumor cells in the center of the tumor (pre- and post-RCT) and invasive front (pre-RCT). k Comparison of ratio of CIC structures, senescent cells and apoptotic cells in the center of the tumor and invasive front compared to frequency of CIC structures in the center of the tumor pre-RCT as a reference
Fig. 2
Fig. 2
Frequency and distribution of CIC structures, senescent and apoptotic cells. a Overall frequency of CIC, senescent cells and apoptotic cells in pre-therapeutic biopsies. Distribution of cell events arranged in order of frequency of (b) CIC, (c) senescence and (d) apoptosis. The percentage of tissue samples having positive events, the overall mean (mean) and mean of tumor tissue with positive events only (mean+) is indicated
Fig. 3
Fig. 3
Correlation analysis. Correlation between (a) CIC and senescence, (b) CIC and apoptosis and (c) apoptosis and senescence
Fig. 4
Fig. 4
Kaplan-Meier analyses. Influence of CIC structures per mm2, senescent cells per mm2 and apoptotic cells per mm2 in the center of the tumor of pre-therapeutic biopsies on NED (a) and overall survival (b). Influence of CIC structures per mm2, senescent cells per mm2 and apoptotic cells per mm2 in the center of the tumor of post-therapeutic biopsies on NED (c) and overall survival (d). The median served as a cut off to separate group in all analyses. Blue solid lines indicate cases below the median and green spotted lines with counts over the median

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