Regulation of Ion Homeostasis for Enhanced Tumor Radio‐Immunotherapy

Abstract Intra/extracellular ion content affects the growth and metastasis of tumor cells, as well as the efficacy of various antitumor therapies. Herein, a carbonic anhydrase inhibitor (CAI) is loaded onto pH‐responsive calcium carbonate (CaCO3) nanoparticles and then modify theses nanoparticles with liposomes to obtain biocompatible CaCO3/CAI@Lipsome (CCL) for enhance tumor radio‐immunotherapy. CCL can specially decompose in tumor microenvironment, releasing calcium ion (Ca2+) and CAI, as well as increasing the pH value of extracellular fluid. CAI restrains the flow of hydrogen ion (H+) inside and outside the tumor cells, resulting in the reversal of tumor acidic microenvironment and the increase of intracellular H+, both of which can improve the sensitivity of tumor to radiotherapy. Afterward, the increased intracellular H+ together with radiotherapy‐causes reactive oxygen species promotes calcium influx, leading to cellular calcium overload. Moreover, the CCL‐tailored content of H+ and Ca2+ strengthens radiotherapy‐induced immunogenic cell death and dendritic cell maturation, amplifying systemic anti‐tumor adaptive immunity. Meanwhile, macrophages in the CCL‐treated tumors are polarized from pro‐tumor M2 to anti‐tumor M1 under X‐ray exposure, owing to the neutralization of tumor acidic microenvironment and enhances Ca2+ content. Therefore, multi‐directional regulation of the intra/extra tumor cell pH/calcium by simple nano‐preparation would provide a powerful way to improve the efficacy of radio‐immunotherapy.

and CAI was 10 mg/kg and 2.5 mg/kg, respectively.These treatments were carried out two rounds.The tumor volume was calculated using a×b 2 /2, and the body weight of the mice was recorded by balance every two days.
The combined therapy of CCL-enhanced radiotherapy and αPD-L1.To evaluate the combined efficacy of CCL-enhanced radiotherapy with αPD-L1, the bilateral CT26 tumor-bearing BALB/c mice were randomly allocated into 4 groups (n = 5) and received treatment as follow: 1) X-rays exposure + surgery; 2) X-rays exposure + surgery + αPD-L1; 3) CCL injection + X-rays exposure + surgery; 4) CCL injection + X-rays exposure + surgery + αPD-L1.The dose of the calcium and CAI was 10 mg/kg and 2.5 mg/kg, respectively, and the dose of X-rays was 6 Gy.The volume of distant tumors and the weight of mice were measured every two days.To analyze orthotropic colon carcinoma inhibition, we first constructed an orthotropic tumor model by injecting 3×10 6 Luciferase-CT26 cells in the mucosa of the colon over the dentate line directly.Then, 10 mice with subcutaneous and orthotropic CT26 tumors were divided into two groups (n = 5) and treated as follow: 1) X-rays exposure+ surgery; 2) CCL injection + X-rays exposure + surgery + αPD-L1.Tumors were monitored by an in vivo imaging System (Lumina III) every 6 days and the weight of mice were measured every two days.
Statistical Analysis.The experimental results were expressed as mean values ± SD.Sample size (n) for each statistical analysis was provided in the figure caption.A two-tailed Student's t-test or a one-way ANOVA was used for the analysis of statistical difference between two groups in GraphPad Prism9.P<0.05 was considered statistically significant.ns, P>0.05; *, P<0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure S2 .
Figure S2.The viability of NIH-3T3 cells incubated with different concentrations of CL, LC or CCL.Data are presented as mean ± s.d.(n = 3).

Figure S4 .
Figure S4.The representative western blotting image showing the expression of CAIX in CT26 cells with indicated treatments.

Figure S10 .
Figure S10.(a) Biodistribution profiles of CL, LC, CCL at 2 h post injection by recording the DiD fluorescence intensity of these homogenized organs and tumors (n = 3).(b) Biodistribution profiles of CL, LC, CCL at 24 h post injection by recording the DiD fluorescence intensity of these homogenized organs and tumors.Data are presented as mean ± s.d.(n = 3).

Figure S11 .Figure S12 .
Figure S11.Biodistribution profiles of Ca 2+ content at 24 h post intravenous injection of PBS or CCL.Data are presented as mean ± s.d.(n = 3).H e a r t L i v e r S p l e e n L u n g K i d n e y T u m o r

Figure S13 .
Figure S13.Tumor volume of primary and distant tumors on the mice in G8 group.Data are presented as mean ± s.d.(n = 5).

Figure S14 .
Figure S14.(a) Body weight curves of mice in various groups.Data are presented as mean ± s.d.(n = 5).(b) H&E-stained major organs in mice with indicated treatment.The scale bar is 200 μm.

Figure S21 .
Figure S21.Body weight curves of mice in various groups.Data are presented as mean ± s.d.(n = 5).