A tumor acidity activatable and Ca2+-assisted immuno-nanoagent enhances breast cancer therapy and suppresses cancer recurrence

Breast cancer recurrence is the greatest contributor to patient death. As the immune system has a long-term immune memory effect, immunotherapy has great potential for preventing cancer recurrence. However, cancer immunotherapy is often limited due to T cell activation being blocked by insufficient tumor immunogenicity and the complex immunosuppressive tumor microenvironment. Here we show a tumor acidity activatable and Ca2+-assisted immuno-nanoagent to synergistically promote T cell activation and enhance cancer immunotherapy. When the immuno-nanoagent reaches the acidic tumor microenvironment, the CaCO3 matrix disintegrates to release immune stimulants (CpG ODNs and IDOi) and Ca2+. CpG ODNs are responsible for triggering dendritic cell maturation to increase the immunogenicity for activation of T cells. And IDOi can inhibit the oxidative catabolism of tryptophan to kynurenine for preventing T-cell anergy and apoptosis. Due to the complexity of the immunosuppressive microenvironment, it is difficult to restore T cell activation by inhibiting only one pathway. Fortunately, the released Ca2+ can promote the activation and proliferation of T cells with the support of the immune stimulants. In vivo experiments demonstrate that our Ca2+-assisted immuno-nanoagent can significantly suppress tumor progression and protect mice from tumor rechallenge due to the long-term memory effect. This immunotherapeutic strategy may provide more possibilities for clinical applications such as treating cancer and preventing relapse.

The flow cytometer assays were carried out with an imaging flow cytometer (Amnis Corporation, USA), and IDEAS image analysis software (Amnis) was used to analyze the images. The optical density (OD value) of was measured with an enzyme labelling apparatus (Thermo scientific, USA). Oxygen consumption rate (OCR) was performed with Extracellular Flux Analyzer (Seahorse bioscience, USA).
CaCO3 nanoparticles were synthesized by means of a gas diffusion reaction. Briefly, 220 mg CaCl2·H2O was dissolved in 100 mL ethanol in a glass bottle covered with aluminium foil, which was punctured with several pores. Then, the bottle was put into a vacuum drying chamber containing 8 g dry ammonia bicarbonate (NH4HCO3). After keeping the whole system in a vacuum environment for 24 h, CaCO3 nanoparticles were obtained and were separated by centrifugation at 12000 rpm. Then, 2 mg CaCO3 was mixed with IDOi in DMSO (2 mg/mL) to obtain CaCO3@IDOi (abbreviated as CaI). The mixture was stirred for at least 24 h to attain maximum loading.
A two-step approach was conducted to modify the above nanoparticles with PEG and PEI. First, 20 mg CaI in ethanol solution and 1 mL DOPA solution (2 mg/mL in chloroform) were mixed under ultrasonication for 20 min. The obtained turbid solution was centrifuged to remove unbound free DOPA and redispersed in chloroform. PEGylation of the nanoparticles was then conducted by mixing a chloroform solution of DPPC, cholesterol and DSPE-PEG at a 4:4:2 M ratio with CaCO3-DOPA under vigorous stirring overnight. Afterwards, the chloroform was evaporated, and the obtained CaCO3@IDOi@PEG nanoparticles were dissolved in aqueous solution for further use.
Then, the obtained nanoparticles were stirred with 200 mg PEI for 24 h to prepare CaCO3@IDOi@PEG@PEI (abbreviated as CaIP). Similarly, CaCO3@PEG@PEI (abbreviated as CaP) nanoparticles were obtained by PEGylation and PEIylation of CaCO3. 5 mg of the above CaIP was stirred with 3OD CpG ODNs in aqueous solution for 12 h to obtain the immuno-nanoagent CaCO3@IDOi@PEG@PEI@CpG (abbreviated as CaIPC).
As a control, the CaP particles were modified with CpG ODNs to obtain CaCO3@PEG@PEI@CpG (abbreviated as CaPC). The prepared nanoparticles were centrifuged at 8000 rpm for 10 min and redispersed in water for further use.

Preparation of liposome@IDOi@PEG@PEI@CpG (LIPC).
For comparison, liposomes loaded with IDOi were prepared by mixing a chloroform solution of DPPC, cholesterol, and DSPE-PEG at a 4:4:2 M ratio with IDOi under vigorous stirring overnight. Afterwards, the chloroform was evaporated, and the obtained liposome@IDOi@PEG nanoparticles were dispersed in aqueous solution for further use. The release profiles of Ca 2+ , IDOi and CpG ODNs.
The release profiles of Ca 2+ , IDOi and CpG ODNs in pH 7.4 and pH 6.5 was studied by ICP-AES, UV/VIS spectrophotometer, and fluorescence spectrophotometer, respectively.

In vivo experiments.
For the xenografts established from cultured cells, 4T1-Luc cells were suspended and harvested after trypsinization, and approximately 5 × 10 5 4T1-Luc cells in 150 μL of serum-free RPMI 1640 medium were subcutaneously injected into the right flank of the mice. The tumor volume (V) was determined by measuring the length (L) and width (W) and was calculated as L × W 2 /2.
To study the antitumor efficacy, mice with tumors were randomly divided into six groups (n = 8) and subjected to different treatments: I, normal saline (NS); II, CaCO3@PEG@PEI (CaP); III, CaCO3@IDOi@PEG@PEI (CaIP); IV, CaCO3@PEG@PEI@CpG To further establish the antitumor performance of CaIPC regarding recurrence, we next used the 4T1-Luc mouse breast cancer incomplete tumor resection model. Mice were randomly divided into six groups (n = 8) and subjected to different treatments after tumors were incompletely resected, leaving behind ~ 1% residual tissue: I, NS; II, CaP; III, CaIP; IV, CaPC; V, LIPC; and VI, CaIPC. Next, the mice in each group were intravenously injected with 50 mg/kg, 5 times in total. The tumor volumes of the mice were measured after 12 days. For comparison, another group of mice were peritumorally injected with 50 mg/kg in each group, 5 times in total.

In vivo bioluminescence and imaging.
Bioluminescence images were collected with an in vivo imaging system (IVIS). The Living Image software (Xenogen) was used to acquire the data 10 min after the intraperitoneal injection of d-luciferin in DPBS (15 mg/mL) into the animals (10 µL/g of body weight).

ELISA analysis.
Serum cytokine levels were determined by enzyme-linked immunosorbent assays (ELISAs) using antibody pairs specific to these cytokines, following protocols Finally, the obtained single cells were stained with fluorescence-labelled antibodies.

Mitochondrial oxygen consumption rate (OCR) of lymphocytes.
The OCR is an important indicator of cellular metabolism and function. In addition, a high OCR indicates a high metabolic rate and good cell viability. To analyse the impact of Ca 2+ on lymphocytes, the OCRs of lymphocytes were studied. The lymphocytes were harvested from the lymph nodes of mice, divided randomly into three groups (n = 3) and subjected to different treatments: i, without treatment; ii, RPMI 1640 with 4 mM Ca 2+ ; and iii, RPMI 1640 with 50 μM BAPTA-AM (a chelator of intracellular Ca 2+ ). The mitochondrial OCR of the lymphocytes was measured 6 h later.

Statistical analysis.
All values in the present study are expressed as means ± SD. The significance between two groups was analysed by a two-tailed Student t-test. P value of less than 0.05 was considered significant (***P<0.001, **P<0.01, *P<0.05).