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. 2022 Jan 14;23(2):877.
doi: 10.3390/ijms23020877.

CD44, γ-H2AX, and p-ATM Expressions in Short-Term Ex Vivo Culture of Tumour Slices Predict the Treatment Response in Patients with Oral Squamous Cell Carcinoma

Pierre Philouze  1   2 , Arnaud Gauthier  1   3 , Alexandra Lauret  1 , Céline Malesys  1 , Giovanna Muggiolu  4 , Sylvie Sauvaigo  4 , Antoine Galmiche  5 , Philippe Ceruse  1   2 , Gersende Alphonse  1   3 , Anne-Sophie Wozny  1   3 , Claire Rodriguez-Lafrasse  1   3
Affiliations

CD44, γ-H2AX, and p-ATM Expressions in Short-Term Ex Vivo Culture of Tumour Slices Predict the Treatment Response in Patients with Oral Squamous Cell Carcinoma

Pierre Philouze et al. Int J Mol Sci. .

Abstract

Squamous cell carcinoma is the most common type of head and neck cancer (HNSCC) with a disease-free survival at 3 years that does not exceed 30%. Biomarkers able to predict clinical outcomes are clearly needed. The purpose of this study was to investigate whether a short-term culture of tumour fragments irradiated ex vivo could anticipate patient responses to chemo- and/or radiotherapies. Biopsies were collected prior to treatment from a cohort of 28 patients with non-operable tumours of the oral cavity or oropharynx, and then cultured ex vivo. Short-term biopsy slice culture is a robust method that keeps cells viable for 7 days. Different biomarkers involved in the stemness status (CD44) or the DNA damage response (pATM and γ-H2AX) were investigated for their potential to predict the treatment response. A higher expression of all these markers was predictive of a poor response to treatment. This allowed the stratification of responder or non-responder patients to treatment. Moreover, the ratio for the expression of the three markers 24 h after 4 Gy irradiation versus 0 Gy was higher in responder than in non-responder patients. Finally, combining these biomarkers greatly improved their predictive potential, especially when the γ-H2AX ratio was associated with the CD44 ratio or the pATM ratio. These results encourage further evaluation of these biomarkers in a larger cohort of patients.

Keywords: CD44; DNA damage response; HNSCC; ex vivo culture; pATM; predictive biomarker; γ-H2AX foci.

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Conflict of interest statement

G.M. and S.S. are employed by the LXRepair Company (La Tronche, France). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
(A). Representative microscopic acquisitions (20×) showing tumour cell proliferation assessed by Ki67 staining in tumour slices after different culture times. (B) Average percentage ± Standard Deviation (SD) of Ki67 tumour nuclei following categories based on the nuclear intensity: grade − (none and weak brown staining); grade + (moderate and strong brown staining). (C) Representative microscopic acquisitions (20×) showing apoptotic tumour cells assessed by TUNEL staining in tumour slices after different culture times. (D) Average percentage ± SD of apoptotic tumour cell nuclei. A minimum of 200 fields per slide were analysed per condition. Student’s t-test was used for statistical analyses (* p < 0.05, ** p < 0.01, **** p < 0.0001).
Figure 2
Figure 2
(A) Representative immunohistochemical (IHC) staining of CD44 expression of responder and non-responder patients in the biopsy sections maintained in ex vivo culture. (B) Mean ± SD of CD44 labelling intensities, quantified using Metafer software (Metasystems, Altlussheim, Germany) for 15 responder and 13 non-responder patients. (C) Ratio of CD44 expression after 4 Gy irradiation compared with 0 Gy for responder and non-responder patients. Two slides and a minimum of 200 fields per slide were analysed per condition. Student’s t-test was used for statistical analyses (ns p > 0.05, * p < 0.05).
Figure 3
Figure 3
(A) Representative microscopic acquisition showing pATM in non-irradiated tumour slices for responders and non-responders. (B) Average percentage ± SD of pATM positive cells 24 h after 0 or 4 Gy irradiation for responder and non-responder patients. (C) Ratio of pATM expression after 4 Gy irradiation compared with 0 Gy for responder and non-responder patients. Two slides and a minimum of 200 nuclei per slide were analysed per condition. Student’s t-test was used for statistical analyses (ns p > 0.05, ** p < 0.01, *** p < 0.001).
Figure 4
Figure 4
(A) Representative microscopic acquisition showing residual γ-H2AX foci in tumour slices of responder patients 24 h after 0 Gy or 4 Gy irradiation. (B) Mean number of γ-H2AX foci ± SD, 24 h after 0 Gy or 4 Gy irradiation for responder and non-responder patients. (C) Ratio of residual γ-H2AX foci after 4 Gy irradiation compared with 0 Gy for responder and non-responder patients. Two slides and a minimum of 400 nuclei per slide were analysed per condition. Student’s t-test was used for statistical analyses (* p < 0.05, ** p < 0.01).
Figure 5
Figure 5
Chronological diagram of biopsy processing.
See this image and copyright information in PMC

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