ICAM-1 orchestrates the abscopal effect of tumor radiotherapy

Abstract

Compelling evidence indicates that radiotherapy (RT) has a systemic inhibitory effect on nonirradiated lesions (abscopal effect) in addition to the ablation of irradiated tumors. However, this effect occurs only in rare circumstances in clinical practice, and mechanisms underlying the abscopal effect of RT are neither fully understood nor therapeutically utilized. Here we identified that intercellular adhesion molecule-1 (ICAM-1), an inducible glycoprotein of the immunoglobulin superfamily, is up-regulated in nonirradiated tumors responsive to RT. ICAM-1 expression in preclinical animal models can be noninvasively detected by optical imaging and positron emission tomography (PET) using near-infrared fluorescence dye- and 64Cu-labeled imaging probes that we synthesized, respectively. Importantly, the expression levels of ICAM-1 determined by quantitative PET imaging showed a strong negative linear correlation with the growth of nonirradiated tumors. Moreover, genetic or pharmacologic up-regulation of ICAM-1 expression by either an intratumoral injection of engineered recombinant adenovirus or systemic administration of a Toll-like receptor 7 agonist-capsulated nanodrug could induce markedly increased abscopal responses to local RT in animal models. Mechanistic investigation revealed that ICAM-1 expression can enhance both the activation and tumor infiltration of CD8+ T cells to improve the responses of the nonirradiated tumors to RT. Together, our findings suggest that noninvasive PET imaging of ICAM-1 expression could be a powerful means to predict the responses of nonirradiated tumors to RT, which could facilitate the exploration of new combination RT strategies for effective ablation of primary and disseminated lesions.

Trial registration: ClinicalTrials.gov NCT04596670.

Keywords: ICAM-1; Toll-like receptor; abscopal effect; positron emission tomography; radiotherapy.

Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.

Identification and validation of ICAM-1 as a biomarker in responsive nonirradiated tumors after RT. (A) Schedule of X-RT in 4T1 bilateral tumor-bearing mice and the definitions of responder and nonresponder mice. (B) Schematic view of proteomic analysis. (C) Grouped scatterplot presenting the change in tumor volume among nonirradiated tumors on day 7 (nonirradiated control tumors, n = 5; nonresponder tumors, n = 8; responder tumors, n = 3). Data are presented as mean ± SD. *P < 0.05, unpaired Student t test. (D) Identified protein numbers in the nonirradiated tumors of the responder group (resp-NIT), nonirradiated tumors of the nonresponder group (nonresp-NIT), irradiated tumors of the responder group (resp-IT), and irradiated tumors of the nonresponder group (nonresp-IT) by proteomic analysis. (E) Numbers of differently expressed proteins in each group. Red represents up-regulated expression, and blue represents down-regulated expression. (F) Heatmap of log2 fold change (normalized to control tumor) of 44 differentially expressed membrane proteins on day 7 in indicated groups. Data represent group mean, n = 3 per group. (Scale bars: white, 0; red, >2 log2 fold change; blue, <2 log2 fold change.) (G) Western blot analysis of ICAM-1 expression in 4T1 tumor tissues on day 7. The densities of Western blot bands were quantified by scanning densitometry with ImageJ software and are presented as the normalized fold change. (H) Immunofluorescence staining of ICAM-1 and Ki67 in 4T1 tumor tissues derived from resp-NIT or nonresp-NIT.

Fig. 2.

Noninvasive imaging of ICAM-1 expression predicts the abscopal response to tumor RT. (A) Schedule of X-RT and in vivo imaging using Dye-αICAM-1/Fab or 64Cu-NOTA-αICAM-1/Fab in the 4T1 or CT26 bilateral tumor-bearing mouse models. (B) Representative in vivo NIRF images and quantitative analysis of tumor uptake of Dye-αICAM-1/Fab at 6 hpi in 4T1 tumor-bearing mice. High or low expression of ICAM-1 was differentiated by quantitative fluorescence intensity (n = 6 to 8 per group). Data are presented as mean ± SD. ***P < 0.001, unpaired Student t test. (C) Representative small animal PET images of 64Cu-NOTA-αICAM-1/Fab at 6 hpi in 4T1 tumor-bearing mice. (D) Growth curves of nonirradiated tumors in 4T1 tumor-bearing mice grouped (ICAM-1 high vs. ICAM-1 low) by quantitative fluorescence intensity in B. (E) Individual growth curves of the irradiated tumors (IT) and nonirradiated tumors (NIT) in the 4T1 tumor model after RT (control group, n = 4; RT group, n = 14). (F) Individual growth curves of the ITs and NITs in the CT26 tumor model after RT (control group, n = 6; RT group, n = 10). (G) Representative small animal PET images of 64Cu-NOTA-αICAM-1/Fab at 6 hpi in CT26 tumor-bearing mice. (H) Growth curves of nonirradiated tumors in CT26 tumor-bearing mice grouped (ICAM-1 high vs. ICAM-1 low) by quantitative fluorescence intensity in SI Appendix, Fig. S13. Tumors are indicated by red circles. *P < 0.05, **P < 0.01, two-way ANOVA (D and H).

Fig. 3.

Expression and functional analysis of ICAM-1. (A) Expression level of membrane ICAM-1 measured by flow cytometry in nonirradiated tumors (n = 6 per group). (B) Assessment of ICAM-1 expression in various cell subsets in nonirradiated tumors. Tumor cells were gated by a CD45− subset and forward scatter. (C) Schematic illustration of X-RT in combination with anti–ICAM-1 blocking or anti-CD8 blocking. (D) Tumor growth curves of irradiated tumors (IT) and nonirradiated tumors (NIT) in 4T1 tumor-bearing mice after the indicated treatments: control (PBS), RT, RT plus anti–ICAM-1 blocking (RT + αICAM-1), and RT plus anti-CD8 blocking (RT + αCD8) (n = 5 per group). (E) Schematic diagram of TAA stimulation and immune synapse formation experiments. (F) Flow cytometry analysis of mutual binding between CD8+ T cells and dendritic cells with or without ICAM-1 blocking (n = 6 per group). (G) Flow cytometry analysis of mutual binding between wild-type or ICAM-1 knockout mouse-derived CD8+ T cells and dendritic cells (n = 4 per group). (H) Flow cytometry analysis of CD107a/IFNγ expression on CD8+ T cells with or without blocking with an anti–ICAM-1 antibody (n = 6 per group). All the numerical data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by one-way ANOVA with Tukey’s post hoc test (A, B, F, and H), two-way ANOVA (D), or an unpaired Student t test (G).

Fig. 4.

Genetic up-regulation of ICAM-1 in vivo by adenovirus enhances abscopal effect of RT. (A) Schematic illustration of X-RT combined with ICAM-1 Ad. (B) Tumor growth curves of irradiated tumors (IT) and nonirradiated tumors (NIT) in 4T1 tumor-bearing mice after the indicated treatments: control (PBS), ICAM-1 expressing Ad alone (ICAM-1 Ad), RT plus PBS (RT + PBS), RT plus control Ad (RT + control Ad) or RT plus ICAM-1 expressing Ad (RT + ICAM-1 Ad) (n = 6 to 9 per group). (C and D) Flow cytometry analysis assessing ICAM-1+ cells (C) and CD8+/CD4+ cells (D) in the nonirradiated tumors after the indicated treatments (n = 5 per group). (E and F) Flow cytometry analysis evaluating the percentage of IFNγ-secreting CD8+ T cells (E) and the ratio of CD8+/Treg cells (F) in nonirradiated tumors of mice after the indicated treatments (n = 5 per group). All the numerical data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 by two-way ANOVA (B) or an unpaired Student t test (C–F).

Fig. 5.

IMQ-induced ICAM-1 up-regulation synergizes with local X-RT to inhibit nonirradiated tumor growth. (A) Schematic illustration of RT combining IMQ and in vivo imaging using Dye-αICAM-1/Fab or 64Cu-NOTA-αICAM-1/Fab. (B) Western blot analysis of ICAM-1 expression in nonirradiated tumor tissues on day 12 after treatment with RT or RT + IMQ. Quantitative results are presented as ICAM-1/β-actin ratio (n = 3 per group). (C and D) Flow cytometry analysis of ICAM-1+ cells and CD8+ T cells (C) and ICAM-1 expression on CD8+ cells and tumor cells (D) in nonirradiated tumor tissues on day 12 after treatment with RT or RT + IMQ (n = 6 per group). (E) Tumor growth curves of nonirradiated tumors in 4T1 tumor-bearing mice after the indicated treatments: control (PBS), IMQ alone (IMQ), RT alone (RT), RT plus anti–ICAM-1 blocking (RT + αICAM-1), RT plus IMQ plus anti–ICAM-1 blocking (RT + IMQ + αICAM-1), and RT plus IMQ (RT + IMQ) (n = 6 to 7 per group). (F) Representative NIRF images and quantitative analysis of tumor uptake of Dye-αICAM-1/Fab at 6 hpi in 4T1 tumor-bearing mice after treatment with RT or RT + IMQ (n = 4 to 5 per group). (G) Representative small animal PET images and quantitative analysis of tumor uptake of 64Cu-NOTA-αICAM-1/Fab at 6 hpi in 4T1 tumor-bearing mice after treatment with RT or RT + IMQ (n = 5 per group). Nonirradiated tumors are indicated by red circles. Numerical data are presented as mean ± SD. (H) Correlation analysis of relative tumor volume on day 20 (V20/V0) and tumor uptake of 64Cu-NOTA-αICAM-1/Fab on day 12. *P < 0.05; **P < 0.01; ***P < 0.001 by unpaired Student t test (B–D, F, and G), two-way ANOVA (E), or Pearson correlation analysis (H).

Fig. 6.

Systemic delivery of IMQ by PLGA nanoparticles enhances abscopal effect of RT in mice. (A) Schematic illustration of the structure of PLGA-IMQ and schedule of X-RT combined with PLGA-IMQ treatment. (B) Tumor growth curves of irradiated tumors (IT) in 4T1 tumor-bearing mice and photographs of tumors harvested at the endpoint (below) after the indicated treatments: control (PBS), PLGA-IMQ alone (PLGA-IMQ), RT alone (RT), and RT plus PLGA-IMQ (RT + PLGA-IMQ) (n = 8 per group). (C) Tumor growth curves of nonirradiated tumors (NIT) in 4T1 tumor-bearing mice and photographs of tumors harvested at the endpoint (below) after the indicated treatments. (D and E) Immunofluorescence staining (D) and quantification (E) of ICAM-1 and Ki67 in nonirradiated tumor tissues after treatment with RT or RT + PLGA-IMQ (n = 5 per group). (F and G) Serial bioluminescence images (F) of 4T1-fLuc tumor-bearing mice after the indicated treatments: RT alone (RT) and RT plus PLGA-IMQ (RT + PLGA-IMQ) (n = 7 to 8 per group). Quantitative results (G) are presented as quantified bioluminescence signals from the lungs. Numerical data are presented as mean ± SD. *P < 0.05; **P < 0.01; ****P < 0.0001 by unpaired Student t test (E) or two-way ANOVA (B, C, and G).