Validation of CD98hc as a Therapeutic Target for a Combination of Radiation and Immunotherapies in Head and Neck Squamous Cell Carcinoma

Abstract

Most patients with head and neck squamous cell carcinomas (HNSCC) are diagnosed at a locally advanced stage and show heterogeneous treatment responses. Low SLC3A2 (solute carrier family 3 member 2) mRNA and protein (CD98hc) expression levels are associated with higher locoregional control in HNSCC patients treated with primary radiochemotherapy or postoperative radiochemotherapy, suggesting that CD98hc could be a target for HNSCC radiosensitization. One of the targeted strategies for tumor radiosensitization is precision immunotherapy, e.g., the use of chimeric antigen receptor (CAR) T cells. This study aimed to define the potential clinical value of new treatment approaches combining conventional radiotherapy with CD98hc-targeted immunotherapy. To address this question, we analyzed the antitumor activity of the combination of fractionated irradiation and switchable universal CAR (UniCAR) system against radioresistant HNSCC cells in 3D culture. CD98hc-redirected UniCAR T cells showed the ability to destroy radioresistant HNSCC spheroids. Also, the infiltration rate of the UniCAR T cells was enhanced in the presence of the CD98hc target module. Furthermore, sequential treatment with fractionated irradiation followed by CD98hc-redirected UniCAR T treatment showed a synergistic effect. Taken together, our obtained data underline the improved antitumor effect of the combination of radiotherapy with CD98hc-targeted immunotherapy. Such a combination presents an attractive approach for the treatment of high-risk HNSCC patients.

Keywords: CD98hc; HNSCC; SLC3A2; biomarker; chimeric antigen receptor; combination therapy; immunotherapy; radioimmunotherapy; radiotherapy.

Figure 1

The combination of the CD98hc-targeted immunotherapies and conventional therapies is a promising therapeutic approach for HNSCC. (A) Conventional therapies (i.e., surgery, radiotherapy, chemotherapy) reduce tumor mass, but tumor relapse can occur. Targeted immunotherapies with universal chimeric antigen receptors T cells (UniCAR) controllable by targeting modules (TM) can be more efficient with fewer side effects. Created with https://biorender.com, accessed on 8 March 2022. (B) Analysis of the TCGA dataset for patients with HNSCC shows an association of the TnB and patient prognosis. (C) Expression levels of TnB negatively correlates with SLC3A2 (CD98hc) gene expression. (D) A combination of the low TnB and high CD98hc identifies a high-risk group of HNSCC patients that might benefit from targeted immunotherapies. In Table S1, we provided a list of 84 genes related to T- and B-cell activation and used for the analyses in Figure 1B–D. TM: Targeting Module; TnB: T- and B-cell activation; UniCAR: universal chimeric antigen receptors T cells.

Figure 2

CD98hc-redirected UniCAR T cells destroy Cal33 RR spheroids. (A) Scheme of procedures for multicellular spheroid formation, UniCAR T treatment, and standard chromium release assay. Created with https://biorender.com, accessed on 31 January 2022. (B) Chromium release assay and flow cytometry analysis showed the efficient elimination of Cal33 RR spheroid cells by UniCAR T cells in the presence of CD98hc TM (50 nM). (C) Representative fluorescence images of Cal33 RR spheroids treated with UniCAR T cells in the presence or absence of CD98hc or EGFR TM. Fluorescence images were taken with Axio Observer Z1 (Zeiss, Jena, Germany). Cal33 RR spheroid: red fluorescence, UniCAR T: green fluorescence. Scale bar: 200 µm. Experiments were performed in triplicates. Paired or nonpaired t-test were applied to calculate the statistical significance of the treatment efficacy (treated vs. control spheroids); error bars, mean ± SEM. * p < 0.05, ** p < 0.01. Cr: Chromium; EGFP: Enhanced green fluorescent protein; E:T: Effector-to-target ratio; NS TM: Nonspecific target module; n.s.: not significant.

Figure 3

Different CD98hc TM concentrations increase UniCAR T cell infiltration into the tumor spheroids. (A) UniCAR T cells were cocultured with Cal33 RR spheroids in the presence of different CD98hc TM concentrations (E:T = 5:1). Half-maximal effective concentration (EC₅₀) value was calculated relative to untreated control based on the obtained dose-response curves. (B) Immunohistochemical analyses of the median sections of spheroids showed increased infiltration levels of UniCAR T cells in the presence of CD98hc TM at a concentration of 250 pM. Infiltrated CD3⁺ T cells (brown spots) were counted blindly by three independent investigators. (C) Immunohistochemical analyses of the median sections of spheroids showing increased infiltration levels of UniCAR T cells in the presence of CD98hc TM at a concentration of 50 nM. Infiltrated CD3⁺ T cells were counted blindly by three independent investigators. Scale bar: 50 µm. Experiments were performed in triplicates. Paired t-test was applied to calculate the statistical significance of the infiltration rate (treated vs. control spheroids); error bars, mean ± SEM. * p < 0.05.

Figure 4

The combination of RT with immunotherapy has a synergistic effect. (A) Scheme, and timeline of procedures for combination therapy. Created with https://biorender.com, accessed on 31 January 2022. Upon spheroid formation, spheroids were given 2 × 2 Gy fractionated irradiation in 2 consecutive days. 6 h after final fraction, UniCAR T treatment was performed in the presence or absence of CD98hc TM. 48 h after treatment, viable cell percentages were analyzed by flow cytometry. (B) RT in combination with CD98-redirected UniCAR T treatment was significantly more efficient in eliminating Cal33 RR and FaDu spheroids than immunotherapy or RT alone. Experiments were performed in triplicates. One-way ANOVA with post hoc Tukey multiple comparison test was applied to calculate the statistical significance of the treatment efficacy (treated vs. control spheroids); error bars, mean ± SEM. * p < 0.05, *** p < 0.001. All calculated p-values are shown in Figure S5A,B. RT: Radiotherapy.

Figure 5

Representative fluorescence images of Cal33 RR spheroids treated with UniCAR T cells in the presence or absence of different CD98hc TM concentrations showing the activation and increase in the EGFP signal of UniCAR T cells in both immunotherapy alone or combination conditions. Fluorescence images were taken with Axio Observer Z1 (Zeiss, Jena, Germany). Cal33 RR spheroid: red fluorescence, UniCAR T: green fluorescence. Scale bar: 200 µm.

Figure 6

Granzyme B and IFN-γ production of CD98hc-redirected UniCAR T cells. UniCAR T cells were cocultured with Cal33 RR spheroids at an E:T ratio of 5:1 together with CD98hc TM at a concentration of 250 pM. (A) After 48 h, UniCAR T cells were analyzed by flow cytometry for intracellular granzyme B expression. Histograms show the UniCAR T cells positive for granzyme B, % (left panel): fluorescence minus one (FMO) control (black line), immunotherapy (red line), RT combined with immunotherapy (blue line). The bar graph shows the median fluorescence intensity (MFI) of granzyme B stained UniCAR T cells (right panel). CAR T cell utilize granzyme B production and can regulate stromal cells via IFN-γ secretion to mediate tumor cell elimination (lower panel). Created with https://biorender.com, accessed on 24 January 2022. (B) IFN-γ production profile of UniCAR T cells incubated with or without Cal33 RR spheroids in the presence or absence of 250 pM CD98hc TM (E:T = 5:1). After 48 h, cell-free supernatants were analyzed by ELISA for IFN-γ. IT: Immunotherapy; RT: Radiotherapy; IFN-γ: Interferon-gamma Experiments were performed in triplicates. One-way ANOVA with post hoc Tukey multiple comparison test was applied to calculate the statistical significance (treated vs. control spheroids); error bars, mean ± SEM. ** p < 0.01, *** p < 0.001.