2.1 Formulation screening for ICG-loaded nanoparticles

To develop an ideal gene delivery vector, formulations of the nanoparticles were optimized using a single-factor screening method with the particle size, encapsulation efficiency (EE), drug loading (DL) capacity, and lipid utilization efficiency (LUE) as indicators.

First, three methods were used to prepare ICG vectors. Nanoparticles1 loaded with ICG (NP1-ICG) (liposome) was prepared by the classical thin-film dispersion method. A two-step method was adopted to prepare lipid-coated ICG-loaded PLGA nanoparticles with shell-core structure as reported previoulsy³⁶ (nanoparticles2 loaded with ICG [NP2-ICG]). Lipid-coated PLGA nanoparticles can also be prepared by one-step method.³⁷

The prescriptions screened for NP1-ICG are shown in Supporting Information: Table S1. ICG is amphiphilic,³⁶ and can be dissolved in both oil phase and aqueous phase during preparation. A higher DL of 8.5% was attained with ICG addition in the aqueous phase relative to the DL of 4.4% with ICG in oil phase, suggesting facile encapsulation of ICG in the aqueous phase of liposome (Supporting Information: Figure S1A,D). In prescription (2)–(5), as the nanoparticle surface charge changed from positive to negative, the EE of ICG decreased correspondingly implying that ICG encapsulation was implemented through electrostatic adsorption between ICG molecule and the phospholipids. In optimized formulation of NP1, the molar ratio of 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP)/L-α-phosphatidylcholine, hydrogenated (Soy) (HSPC)/cholesterol (Chol) was 1/2.8/1.2, and particle size, potential, EE and DL were 149.1 nm, 37.8 mV, 92.6%, and 8.5% (w/w), respectively.

The prescriptions screened for NP2-ICG are shown in Supporting Information: Table S2. With the increase of PLGA/lipid weight ratio, the nanoparticle particle size also increased probably ascribed to the increased size of PLGA inner core.³⁸ The optimum particle size was 163.8 nm, but the EE and DL were only 52.3% and 5% (w/w), respectively. The low EE of ICG was probably ascribed to ultrasonication after ICG loading, which might lead to ICG leakage (Supporting Information: Figure S1B,E).

The prescriptions screened for nanoparticles 3 loaded with ICG (NP3-ICG) are shown in (Supporting Information: Table S3). Rise of polyvinyl alcohol (PVA) concentration resulted in increase of NP3-ICG particle size owing to a decreased shear force during the emulsification process (Supporting Information: Figure S1C,F).³⁹ And higher sonication times might induce an increase in NP3-ICG particle size resulting from demulsification. Prescriptions (8), (9), (11), and (12) were identified as the better prescriptions. Compared with two-step method, NP3-ICG exhibited smaller particle size, higher lipid utilization (Supporting Information: Figure S2A) and DL. The optimal formulation for NP3-ICG was shown in Table 1. In conclusion, the one-step NP3 was selected for further study.

2.2 Characterization of pharmaceutical and biological properties of the nanoparticles

After obtaining the optimized prescription, the main properties of the nanoparticles including size, gene condensation rate. photothermal effect, cytotoxicity, tumor targeting effect in tumor-bearing mice as well as photothermal protransfection effect were comprehensively investigated.

NP3-ICG is a homogeneous sphere with a round-like shape examined by transmission electron microscopy (Figure 1A). NP3-ICG had good stability in terms of particle size, polydispersity coefficient (PDI), and potential measured at 4°C within 14 days (Supporting Information: Figure S2B). The maximum UV absorption peak of NP3-ICG at 805 nm (Supporting Information: Figure S2C) indicated that ICG was effectively loaded onto NP3. The binding ratio of NP3-ICG to red fluorescence protein (RFP) detected by agarose gel electrophoresis (Figure 1B, Supporting Information: Figure S2D) showed that all three nanoparticles completely condensed the plasmid DNA at N/P ratio > 4. Formulation 12 of NP3-ICG at N/P ratio of 10 displayed the best transfection efficiency on both CT26 and 293T (human embryonic kidney cells) cells in vitro relative to formulation 8/9 indicating that the molar ratio of DOTAP was positively correlated with the transfection rate (Supporting Information: Figure S3). And DOTAP might achieve a higher transfection efficiency through enhanced cellular uptake and lysosome escape mediated by cell membrane binding and the proton sponge effect of DOTAP.³⁸ NP1-ICG and NP3-ICG at N/P ratio of 20 resulted in lower transfection efficiency probably imputed to high cytocotoxity (Supporting Information: Figure S3).

The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, Thiazolyl Blue Tetrazolium Bromide) result showed that the half maximal inhibitory concentration (IC50) of NP1-ICG and NP3-ICG were 33.3 and 50 μg/mL, respectively (Supporting Information: Figure S4A). The main organs had no obvious pathological lesions after the administration of NP3-ICG. Liver and kidney function-related indexes such as serum urea, creatinine, alanine transaminase, and glutathione transaminase showed no apparent change (Supporting Information: Figure S4B,C). NP3-ICG did no cause visible hemolysis (Supporting Information: Figure S4D). In summary, NP3-ICG had good biocompatibility and was used in the following research.

After administration of NP3-ICG-DID (1,1-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate as DID) to tumor-bearing mice, the highest accumulation in the tumor site was observed at 6 h, second only to that of liver, suggesting that the vector has tumor targeting capability (Supporting Information: Figure S5A–D). Besides, the fluorescence of DID was also detected in the tumor sections of these mice, of which the fluorescence strength at different time points was consistent with semiquantitative results of tumor nodules (Supporting Information: Figure S5E). The photothermal effect of NP1-ICG and NP3-ICG were comparable with peak temperature reached up to 48°C after PTT irradiation in vitro, which was higher than the free ICG (Supporting Information: Figure S6).^{40, 41} And the photothermal efficiency was augmented with increasing NIR power and ICG concentration. The photothermal effect of the nanoparticles in vivo was also examined (Figure 1G, Supporting Information: Figure S7). The nanoparticles administered intravenously at dose as high as 8 mg/kg of ICG made the temperature at the tumor site rise to around 41°C and lasted 40 min followed by 808 nm laser irradiation. And a lower dose (4 mg/kg ICG) barely caused temperature rise (Supporting Information: Figure S7). By contrast, NP3-ICG after intratumoral injection, exhibited a more significant photothermal effect, which was promoted with increasing both NIR power and ICG dose. And a low dose of 1 mg/kg of ICG with NIR power of 0.6 w/cm² would make the tumor temperature rise to 42°C. As the optimal temperature range for inducing HSP70 expression was 39–43°C and temperature over 43°C might cause cell death,¹⁵ the final irradiation conditions were set as intratumoral administration of NP3-ICG (ICG at 1 mg/kg) with NIR power of 0.6 w/cm².

In this paragraph, the promotion of NP3-ICG endocytosis and transfection in vitro by photothermal effect will be discussed. It has been reported in several studies that the photothermal effect exhibited pleiotropic effects on promoting transfection through enhanced cellular uptake of the vector, accelerated lysosome escape for gene cytoplasmic delivery, and induced gene release from the vector. To obtain the optimal photothermal effects, ICG concentration and irradiation time points after vector administration were optimized in 293T and CT26 cells. The screening of irradiation time point indicated that irradiation right after transfection gave the highest transfection efficiency, which was probably because the impact of the photothermal effect on nanoparticle endocytosis overwhelmed those on nanoparticle lysosome escape and gene cytoplasmic release. Both too high NIR power and ICG concentration would cause cell death, which explained the reduction of gene transfection efficiency when these two factors were at too high level. The optimal PTT condition after screening was irradiation at 0 h with ICG concentration of 6 μg/mL and NIR power of 0.6 W/cm² (Supporting Information: Figure S8,9). Photothermal effect on the optimized irradiation condition remarkably enhanced vector endocytosis and RFP expression in both cell lines (Figure 1C–F, Supporting Information: Figure S10A). NP3-ICG+NIR group induced a 14.8-fold higher luciferase expression with an HSP70 promoter compared to the control group with NP3-ICG transfection only (Figure 1G,H, Supporting Information: Figure S10B).

It was worth noting that NP3-ICG+PTT group showed a 7.9-fold increase in luciferase expression relative to NP3-ICG+hyperthermia group. Compared with the hyperthermia effect of water bath treatment, PTT enabled superior gene expression through multiple effects as described above.

2.3 Antitumor effect in vitro

Chemotherapy drugs with a caspase-dependent apoptosis inducing effect can trigger pyroptosis by cleaving gasdermin proteins with activated caspase 3, thus converting apoptosis into pyroptosis. OXA can initiate apoptosis by activating caspase-3. Whether OXA can cleave GSDME in tumor cells and consequently induce pyroptosis is not determined, which will be investigated in this section.

The results showed that the infection rate of CT26 and HCT116 were 85.4% and 78.7%, respectively (Figure 2C, Supporting Information: Figure S11A,B). G-CT26 and G-HCT116 cell mortality rate after OXA treatment increased about 20.9% and 13.4% compared to control group (Figure 2C, Supporting Information: Figure S11B).

Cleavage of caspase-3 and GSDME occurred after OXA treatment in both CT26 and HCT116 cells with overexpression of GSDME, which explains the variation in total protein expression after OXA treatment (Figure 2A,B, Supporting Information: Figure S12A,B). The result was consistent with previous report that activated caspase-3 induced by chemotherapy drug like OXA would cleave GSDME, thereby realizing antitumor mechanism transformation from apoptosis to pyroptosis.

As calreticulin (CRT) and high mobility group box 2 (HMGB2) are representative DAMPs and in favor of antitumor immunity, their expression in tumor tissues were studied by immunohistochemistry assay (Figure 2E,F). CRT expression after OXA treatment was higher in G-CT26 than CT26; on the contrary, HMGB2 expression inside of G-CT26 was diminished more strikingly than that in CT-26. Studies have demonstrated that CRT is overexpressed on tumor cell surface after drug treatment such as anthracyclines. CRT can enhance antitumor immune responses by binding to CD91/LRP1, a receptor located on the surfaces of innate immune cell.⁴² Meanwhile, secreted CRT can not only control microvessel growth but also prevent tumor cell reproduction. In normal physiological conditions, HMGB2 is mainly located in nucleus, while it is secreted outside of the cells at the stage of necroptosis or even in the late stage of apoptosis causing inflammatory reactions. These results suggested that overexpression of GSDME would enhance the immunogenicity of CT26 cells after OXA treatment.

2.4 Antitumor effects in vivo and analysis of immune cell population variation in tumor microenvironment

As discussed above, OXA not only mediated caspase-3-dependent GSDME cleavage, but also induced DAMPs release. Whether OXA in combination with GSDME-pDNA loaded nanoparticles would play a tumor-suppressing effect in vivo and simultaneously activate intense antitumor immunity was discussed in this section using xenograft, orthotopic as well as distal tumor models. The feasibility of GSDME-overexpressing CT26 cells treated with OXA as a tumor vaccine were also explored.

In preliminary experiment, mG-NP3-ICG administration route and irradiation duration were first screened in preliminary experiments with tumor-bearing mice (Supporting Information: Figure S13A,B). The tumor suppression effect was superior when the irradiation lasted for 30 min. Although the tumor inhibitory rate was comparable between intratumoral and intravenous administration groups, the intratumoral group would not cause obvious body weight loss of mice. Based on these results, intratumoral administration and irradiation for 30 min were employed in the subsequent experiments.

In the xenograft colon-cancer model (Figure 3), the highest tumor inhibition ratio reached 77.7% in the mG-NP3-ICG+PTT+OXA group without significant changes in body weight of mice. And the survival period was also the longest compared with all other groups. As shown in hematoxylin and eosin staining sections (Supporting Information: Figure S14), no significant organ injury occurred in all groups, while apparent necrosis occurred at tumor site in the mG-NP3-ICG+PTT+OXA group. And this group showed a remarkable decrease in neovascularization (CD31) at tumor sites as well as the quantity of proliferative tumor cells (KI67). Besides, CRT was also conspicuously increased, while HMGB2 was decreased at the tissue level (Supporting Information: Figure S15–17).

In the distal tumor model, although OXA has been reported to induce immunogenic cell death, the inhibitory effect of OXA or mG-NP3-ICG+PTT alone on either the primary or distal tumor was limited in the CT26 distal tumor model. In contrast, antitumor effect of the mG-NP3-ICG+PTT+OXA group was not affected by inoculation of distal tumors. And the tumor inhibition efficiency reached 84.5%, which is similar to the result of xenograft CT26 model (Figure 4). It deserves to be mentioned that mG-NP3-ICG+PTT+OXA group also displayed definite antitumor effect on distal untreated tumors, implying that the combination group indeed enhanced the antitumor immunity and thus remarkably inhibited the untreated tumors with the potential to arrest distal tumor metastasis. No visible weight loss of the mice was observed in all groups during the whole treatment process indicating good tolerance (Figure 4D).

In the orthotopic CT26 tumor model (Supporting Information: Figure S18), the inhibition efficiency of mG-NP3-ICG+NIR+OXA group for the subcutaneous tumor was 85% and was comparable to those of the xenograft and distal tumor models, which was also not affected by the intraperitoneal inoculation of tumors. And tumor inhibition rates for the orthotopic tumors of OXA, mG-NP3-ICG+NIR, and mG-NP3-ICG+NIR+OXA groups were 40%, 5.4%, and 48.8%, respectively. In our experimental procedure, tumors were inoculated both subcutaneously and intraperitoneally at the same time. After tumor inoculation, the treatment was started on the 10th day when the subcutaneous tumor was big enough for the intratumoral injection and irradiation. Nevertheless, the peritoneal tumors growed so rapidly that it was difficult for the intervention started on 10th day to control their growths, which probably explained the low inhibition rate in our study.

Insufficiency in lymphocyte infiltration at the tumor site in CRC is a major cause of poor antitumor efficacy for chemotherapeutic agents, so the effects of various treatments on tumor-infiltrating lymphocyte (TIL) subpopulations were systematically examined (Figure 5). We analyzed the change in proportions of NK cells, CD11b+ cells, and CD8+ T cells. Although the proportion of these subsets increased in all treatment groups, the change in the combination group was the most significant, with raised CD45+ cells, NK cells, CD11b+ cells, and CD8+ T cells of 23.41%, 11.64%, 15.77%, and 6.78%, respectively.

Antitumor vaccine is the gold standard for validating drug-induced immunogenic cell death. According to Figure 6, mice inoculated with G-CT26 after treatment with OXA was effective in preventing tumorigenesis with tumor suppression efficiency of 89% and had no apparent loss in body weight. These results were consistent with literature, suggesting that the combination group had the potential as an antitumor vaccine.⁴³

5.1 Reagents and plasmids

Indocyanine green was purchased from MedChemExpress. Oxaliplatin (OXA, 98%) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. DOTAP, 1,2-dimyristoy-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA), HSPC, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) were purchased from Lipoid. Chol was purchased from Shanghai Bio Science & Technology. PLGA (lactic acid:glycolic acid = 50:50) was purchased from Shandong Institute of Medical Instruments. PVA (Molecular weight  = 30,000–50,000, 87%) was purchased from Sigma-Aldrich. DID (>98%) was purchased from Biotium. Coumarin 6 (C6, >98%) was purchased from TCI Development Co., Ltd. 4′,6-diamidino-2-phenylindole (DAPI) was purchased from J&K Scientific. Propidium iodide (PI) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. Annexin V-APC (550475) was purchased from BD Pharmingen. CD31(GB11063-3) and KI67(GB111499) was purchased from Servicebio. CRT(12,238 s), caspase-3(9665), and p-p53(9284) were purchased from Cell Signaling Technology. HMGB2(ab67282) was purchased from Abcam. Anti-GAPDH(AF5718) was purchased from R&D. DFNA5(K2217) was purchased from Santa Cruz Biotechnology. FlowAB-AF555(A21429), FlowAB-AF488(A10667), and PEcy7-NK1.1(25-5941-82) were purchased from ThermoFisher. FITC-CD45.2(09806) and PB-CD11b(101224) was purchased from biolegend. PEcy7-CD8(25-0081-81) was purchased from Invitrogen.

RFP expressing reporter plasmid was purchased from Invitrogen. pDRIVELucia-mHSP70 was purchased from Invivogen. pcDNA3.1-mHSP70-mGSDME and pcDNA3.1-hHSP70- mGSDME were constructed by GENEWIZ.

5.2 Cell culture and animals

Mouse 293T cells were purchased from Nanjing Kebai Biotechnology Co., Ltd. and cultured in DMEM supplemented with 10% fetal bovine serum together with 1% penicillin/streptomycin and incubated at 37°C, 5% CO₂. Mouse CT26 cells and human HCT116 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd. and cultured in 1640 containing 10% fetal bovine serum and 1% penicillin/streptomycin, and incubated at 37°C, 5% CO₂.

Male Balb/c mice (6–8 weeks old, 20 ± 2 g) were purchased from Sichuan University Experimental Animal Centre and fed in the purified air laminar flow frame of the Animal Center of the State Key Laboratory of Biotherapy, Sichuan University. All animal experiments involved in the present study were approved by the Animal Care and Use Committee of Sichuan University.

5.3 NP3-ICG preparation

An oil phase containing PLGA/lipids (10 mg) in dichloromethane (DCM) with a bulk volume of 0.48 mL containing a total material mass of 10 mg was emulsified with 0.16 mL of an internal aqueous phase containing ICG solution (1 mg) for 1 min (5 s on/5 s off) to obtain the W/O primary emulsion using a probe ultrasonicator. The primary emulsion was further emulsified with an outer aqueous phase containg 1.8 mL of 1% PVA also using the probe ultrasonicator for 1 min to attain the W/O/W double emulsion. The nanoparticle suspension after removal of organic solvent under vacuum at 37°C was centrifuged at 13,300 rpm for 90 min at 4°C to obtain NP3-ICG in the precipitate. EE and DL efficiency of ICG were determined by detecting the absorbance of unencapsulated ICG at 780 nm in the supernatant after centrifugation through a UV spectrophotometer (UV-1750; Shimadzu). And EE and DL efficiency were calculated by the following equitations, in which C_t represents the initial amount of drug added and Ce represents the amount of encapsulated drug. DID-labeled NP3 (DID-NP3-ICG) was prepared as described above with the addition of DID in the oil phase at a content of 0.25% (w/w).

C6-NP3-ICG was prepared following the preparation of C6-NP2-ICG described in the attachment, and LUE was calculated accordingly.

5.4 Characterization of size, zeta potential, and morphology

The particle size, zeta potential, and PDI were determined by Malvern Nano-ZS 90. Then the morphology of the nanoparticles was visualized by transmission electron microscope (TEM) (H-600; Hitachi) following negative staining using 2% phosphotungstic acid. The stability of the nanoparticles stored at 4°C was determined after 14 days.

5.5 Integrity of pDNA

The nanoparticles dispersed in deionized water were incubated with the reporter gene at various N/P ratios from 1 to 40. And the binding ratio was evaluated by agarose gel electrophoresis at 120 V for 20 min.

5.6 Photothermal effect of the nanoparticles in vitro and in vivo

The nanoparticles were irradiated with an 808 nm laser (Changchun Leishi Photo-Electric Technology Co., Ltd.) and the temperature variation of the nanoparticle suspension within 40 min was measured consecutively.

Mouse was administered with the NP3-ICG intravenously. After 24 h, the xenograft tumors were irradiated with NIR (808 nm laser). The temperature change at the tumor site was measured consecutively over 40 min.

The temperature variation was visualized by an Infrared Thermal Imager (H36; Hikvision).

5.7 Transfection of the nanoparticles in vitro

293T or CT26 (5 × 10⁴) cells were seeded in 24-well plates. After 24 h, the cells were transfected with NP3-ICG loaded with RFP-expressing pDNA or pDRIVE5Lucia-mHSP70 followed by irradiation with 808 nm laser and incubated with the transfection reagents for 4 h before the medium was replaced with fresh medium. At 48 h after transfection, RFP expression was observed by a fluorescent microscope (OLYMPUS). The secreted luciferase encoded by pDRIVE5Lucia-mHSP70 in the supernatant utilizes the coelenterazine as substrate for luciferase reaction. And the light signal produced can be quantified using a fluorescent plate reader (Biotek) and expressed as relative light units (RLU).

5.8 MTT assay

CT26 cells (1 × 10⁴) were seeded in 96-well plates and cultured for 24 h. NP3-ICG with various concentrations of ICG in the media (5, 12.5, 25, 50, 125, 250, 500, 1000, and 2000 µg/mL) were used to treat the cells for 48 h. Then, 20 µL of MTT solution (5 mg/mL, pH 7.4) was added to each well and incubated for 4 h. The formazan crystals formed were dissolved in dimethyl sulfoxide (150 µL/well), and absorbance was read at 490 nm on a UV spectrophotometer. The cell viability was positively correlated with the amount of formazan crystals.

5.9 Biodistribution in vivo

NP3-ICG-DID (DID 0.5 mg/kg) was slowly infused via the tail vein and biodistribution of the nanoparticles in vivo was observed by an in vivo imaging system (Perkin Elmer).

5.10 Flow cytometry assay for analyzing cell viability and change of TIL subtypes

Tumor cells stably expressing human/mouse GSDME were treated with various antitumor drugs for 24 h, of which the apoptosis markers were detected by flow cytometry after staining with Annexin V/PI (BD Biosciences).

Tumors resected from the treated or control mice were digested with collagenase D (1 mg/mL; Sigma-Aldrich) and DNase I (0.05 mg/mL; Sigma-Aldrich) for 40 min at 37°C followed by passing through 70 μm cell strainer. The TILs were isolated by percoll and stained with anti-CD45-FITC, anti-CD11b-PB, anti-CD8-PEcy7, and anti-NK1.1-PEcy7 before subjecting to flow cytometry analysis.

5.11 Western blot

Cells or tissues were harvested and lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktails. Bicinchoninic acid (BCA) protein assay (Beyotime Biotechnology) was used to determine protein concentration. Then equal amounts of proteins were separated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred onto the Nitrocellulose membrane. The transferred membranes were blocked with 5% skim milk at room temperature, then incubated with primary antibodies including anti-caspase-3 (9665; CST), anti-DFNA5 (sc-393162; SANTA), and anti-GAPDH (AF5718; R&D) at 4°C overnight. Subsequently, the membranes were incubated with the horseradish peroxidase secondary antibody at room temperature and developed by ECL (Beyotime Biotechnology).

5.12 Immunofluorescence

Treated cells were fixed in 4% paraformaldehyde solution for 10 min. After permeabilization with 0.1% Triton X-100 PBS for 10 min, they were washed three times with PBS and blocked with 5% bovine serum albumin for 30 min. Then, the cells were incubated with primary antibody like anti-CRT and anti-HMGB2 at 4°C overnight followed by incubation with fluorescent probe-labeled secondary antibodies and counterstaining with DAPI solution. Finally, the slides were visualized with a confocal microscope (Zeiss 880).

5.13 Immunohistochemistry

Frozen sections of the tumors were treated as described in Section 5.12 except for different primary antibodies used including anti-CD31, anti-KI67, anti-CRT, and anti-HMGB2. The sections were visualized with a fluorescent microscope (Olympus IX-70).

5.14 Xenograft colon cancer model

GSDME-pDNA-loaded NP3-ICG abbreviated as mG-NP3-ICG was prepared by incubating NP3-ICG with GSDME-pDNA at a N/P ratio of 10 and used for the following experiments. Female Balb/c mice were inoculated subcutaneously with CT26 (1 × 10⁶ cells/each mouse) and randomly grouped on Day 10. The treatment groups included (a) model group without treatment (control group), (b) OXA (i.p., 5 mg/kg), (c) mG-NP3-ICG+NIR (i.t., ICG 1 mg/kg), (d) mG-NP3-ICG+NIR+OXA (mG-NP3-ICG, i.t., ICG 1 mg/kg; OXA, i.p., 5 mg/kg). At 12 h after intratumor administration of the nanoparticles, the tumors were irradiated with NIR followed by intraperitoneal administration of OXA after 24 h. The treatment was repeated on Day 13 and Day 16. The mice were killed on Day 20, then mice weight, volume (V = length × wenth² × 0.5) and weight of tumor were measured. Main organs were resected for further study.

5.15 Distal colon cancer model

CT26 (2 × 10⁶) was inoculated subcutaneously on both sides of hind limbs of mice. Tumor treatment and tumor inhibition study were performed as described in Section 5.14 on the 10th day.

5.16 Orthotopic colon cancer model

CT26 was inoculated both subcutaneously (2 × 10⁶) and intraperitoneally (1 × 10⁵) in mice. And only the subcutaneous tumors were treated. Tumor treatment and tumor inhibition study were performed as described in Section 5.14.

5.17 Antitumor vaccines

Mice were immunized twice with CT/G-CT26 after treatment of OXA for 24 h as vaccines on left hind limb on Days 14 and 7, and were rechallenged with CT26 (2 × 10⁶) on right hind limb on Day 0. Tumor volume and mouse weight were assessed afterward.