Synthesis and characterization of the CR NPs
First, the D–π–A–π–D structured CR-TPE-T was synthesized by a single-step condensation between croconic acid and N, N-bis(4-(1,2,2-triphenylvinyl) phenyl) thiophen-2-amine in 1:2 equal primarily based on the earlier studies [32] (Scheme S1). The molecular geometry and digital distribution of CR-TPE-T have been studied by density useful concept (DFT) calculations. As proven in Fig. 1a, the optimized ground-state (S0) geometry of CR-TPE-T confirmed an axisymmetric configuration in house; the 2 dihedral angles between TPE and its neighboring thiophene ring have been 52.9° and 44.6°. The dihedral angle between the thiophene ring and the croconic ring was 1.0°, and the entire molecule tended to look as a planar conformation alongside the spine. In CR-TPE-T, the electron densities of the best occupied molecular orbital (HOMO) have been effectively distributed on the tetraphenylethylene (TPE) donors alongside its conjugated thiophene items, whereas the bottom unoccupied molecular orbital (LUMO) was extra delocalized on the electron-deficient croconaine core, revealing environment friendly intramolecular cost switch throughout the molecule. The HOMO and LUMO power bandgap of CR-TPE-T was roughly 1.45 eV (Fig. 1b).
Characterization and properties of the CR NPs. (a) Chemical construction and optimized molecular geometry of CR-TPE-T. (b) The calculated frontier molecular orbitals of CR-TPE-T calculated by the density useful concept (DFT) calculation methodology on the B3LYP/6-31G degree. (c) Absorption and fluorescent spectra (excited wavelength 808 nm) of croconaine dye nanoparticles (CR NPs) in aqueous resolution. (d) Consultant transmission electron microscopy (TEM) picture and (e) Hydrodynamic dimension distribution of CR NPs. scale bar: 200 nm. Polydispersity index (PDI) = 0.15. (f) Dimension of CR NPs after 5 weeks of storage at nighttime at room temperature
For in vivo functions, the hydrophobic CR-TPE-T was then fabricated into nanoparticles (NPs) (CR NPs) by nanoprecipitation utilizing DSPE-mPEG2000 because the encapsulation matrix. Based on the normalized absorption spectrophotometer evaluation, the calculated encapsulation effectivity of the CR NPs was 89% (Determine S1). The UV-vis-NIR absorption spectra evaluation of the CR NPs displayed an intense attribute absorption peak of 870 nm and increasing over 1000 nm, matching very effectively with the organic window (700–1000 nm), which means that it has potential to function a PTA. As well as, the CR NPs confirmed the corresponding fluorescence emission overlaying the NIR-II area, which extends to 1200 nm, with most peak at 970 nm, the fluorescence quantum yield of CR NPs in water was calculated to be 0.47% through the use of IR 1061 as a reference (Determine S2), indicating the potential for NIR-II FLI (Fig. 1c).
The TEM picture and DLS leads to Fig. 1d and e indicated a dispersed spherical form of CR NPs with common diameter of ∼ 158 nm, indicating that the NPs may obtain passive tumor accumulation by the EPR impact. Moreover, the CR NPs zeta potential was − 33.7 ± 1.71 mV, which favors good stability in a physiological setting (Determine S3). The colloidal stability of CR NPs was additional studied by recording their particle dimension at room temperature for five weeks. Notably, negligible variation of the diameter distribution was noticed for the NPs resolution after storage at room temperature for five weeks, implying good colloidal stability of the CR NPs (Fig. 1f). These outcomes show that CR NPs are a promising candidate for NIR-II FLI-guided PTT functions.
In vitro NIR-II FLI and PTT properties of CR NPs
Due to the inherent NIR absorption capability of the CR NPs, we then investigated the PTT habits of CR NPs in vitro. As proven within the infrared photos (Fig. 2a) recorded by a thermal digital camera, the CR NPs exhibited an obvious photothermal impact on 808-nm laser irradiation. The laser energy density results on the temperature modifications of CR NPs have been then investigated. It was evident that the profiles of temperature increments of the CR NPs have been positively associated to laser energy density (Fig. 2b). The temperature elevated to roughly 62.5 °C on the energy density of 1 W cm− 2 on 808-nm laser irradiation for six min, which was sufficient to kill tumors successfully. The CR NPs focus (25 to 200 µg mL− 1) additionally confirmed a optimistic relationship with the photothermal impact at 1 W cm− 2 of laser energy density and the utmost temperature was 64.3 °C (Determine S4b), whereas the management (phosphate-buffered saline [PBS]) group confirmed a negligible temperature change below equivalent situations. The corresponding infrared (IR) thermographs confirmed the temperature modifications (Fig. 2c and S4a). These outcomes point out controllable photothermal habits. Photothermal stability is a crucial parameter for evaluating the potential of PTA. Then, the CR NPs have been uncovered to 808-nm laser over 5 steady irradiation and cooling cycles to guage its photothermal stability (Fig. 2d). The best temperature of the CR NPs in every cycle was remarkably constant, even after 5 cycles of irradiation, implying the excellent thermal- and photo-stability. Moreover, on foundation of the cooling curve (Fig. 2e) and the reported strategies [43, 44], the estimated PCE of the CR NPs was 65%. To discover whether or not CR-NPs exhibit photodynamic results, we studied the ROS manufacturing functionality of CR NPs below 808 nm laser publicity. We used 1,3-diphenylisobenzofuran (DPBF) as an extracellular 1O2 trapper as a result of it may be irreversibly oxidized by 1O2 [45]. As proven in Determine S5, the absorption depth didn’t considerably lower after laser irradiation, indicating no 1O2 was generated and, consequently, that CR-NPs don’t exhibit photodynamic results. Subsequently, CR-NPs primarily operate by photothermal results with out concurrent photodynamic exercise.
In vitro NIR-II FLI and PTT properties of CR NPs. (a) Infrared (IR) thermal photos of 200 µg mL–1 CR NPs aqueous resolution below 5 min irradiation (808 nm, 1.0 W cm− 2) adopted by 5 min cooling interval. (b) Photothermal efficiency and (c) real-time thermal imaging of CR NPs in aqueous resolution with different laser energy densities (200 µg mL–1, 808 nm). (d) Photothermic stability of 200 µg mL− 1 CR NPs in aqueous resolution (808 nm, 1.0 W cm− 2) throughout 5 successive laser ON/OFF cycles. (e) Time versus − Ln(θ) linear correlation derived from the cooling stage of Fig. 2d. (f) In vitro NIR-II alerts of aqueous resolution of CR NPs at totally different concentrations (0.1 − 0.5 mg mL− 1) below 808-nm excitation. (g) Quantitative relationship between fluorescence imaging (FLI) sign intensities and concentrations of CR NPs
Motivated by the excellent optical efficiency of the CR NPs, we then studied the CR NPs’ in vitro NIR-II FLI efficiency utilizing a 900-nm long-pass (LP) filter below 808-nm laser excitation. As depicted in Fig. 2f and g, the CR NPs confirmed robust NIR-II fluorescence in a concentration-dependent method, with the fluorescence sign linearly correlated with its focus within the vary of 10 to 50 µg mL− 1, suggesting the feasibility of NIR-II bioimaging.
In vitro antitumor efficiency analysis of CR NPs
Given the superior photothermal properties of CR NPs, its PTT effectiveness in Colon26 and 4T1 was then studied (Fig. 3 and Determine S6). First, the photocytotoxicity of CR NPs was studied utilizing a typical CCK-8 cell viability assay. As proven in Fig. 3a, with out laser irradiation, the cell viability for Colon26 was negligibly influenced even the focus of CR NPs was as much as 40 µg mL− 1, revealing the superb biocompatibility of CR NPs for biomedical and scientific functions. In distinction, vital photothermal cytotoxicity was noticed after therapy with CR NPs + laser irradiation, and the cell viability decreased markedly with greater than 80% of cells dying at a CR NP focus of 20 µg mL− 1. Related outcomes have been additionally obtained in 4T1 cells, displaying that CR NPs + laser irradiation might considerably suppress the cell viability in a concentration-dependent method (Determine S6a). In the meantime, we seen that 4T1 cells seemed to be extra delicate to CR NPs-induced photothermal impact than Colon26 since decrease concentrations of CR NPs akin to 2.5 and 5 µg ml− 1 have prompt distinguished inhibition to viability upon irradiation. To additional examine the photothermal impact of CR NPs, we then carried out stay (inexperienced fluorescence)/useless (crimson fluorescence) staining experiments. As proven in Determine S6b, cells handled with PBS, CR NPs and PBS + laser irradiation exhibited widespread inexperienced fluorescence, indicating that there was no anticancer impact in these teams. Purple fluorescence was clearly noticed within the CR NPs + laser irradiation group, and nearly 70% of cells underwent dying after irradiation. This illustrates the superb PTT capability of CR NPs towards Colon26 cells, which is in keeping with CCK-8 findings. Moreover, cell apoptosis was studied by stream cytometry. As anticipated, the CR NPs + laser group confirmed a remarkably excessive cell apoptotic ratio (∼95%), whereas, within the management teams, no apparent apoptotic or necrotic cells have been noticed, which additional confirmed the excessive effectivity of the PTT impact of CR NPs (Fig. 3b and c). Collectively, CR NPs confirmed good biocompatibility and environment friendly PTT below laser irradiation, implying nice potential for in vivo biomedical functions.
In vitro antitumor efficiency analysis of CR NPs. (a) Relative Colon26 cell viability in relation to numerous CR NPs concentrations with or with out laser irradiation, decided utilizing a CCK-8 assay (n = 6). (b) Apoptosis evaluation on Colon26 cells utilizing stream cytometry after totally different remedies (CR NPs: 20 µg mL− 1). For (a) and (b), laser irradiation situations: 808 nm, 1.0 W cm− 2, 5 min. (c) The corresponding quantitative evaluation of (b). (d) Immunofluorescence staining of cell floor CALR and HMGB1 expression in numerous teams (n = 4). Scale bars = 50 μm
Research have indicated that photothermal ablation of tumors might stimulate and redistribute immune cells, thereby triggering strong antitumor immune responses [46]. An important facet of this immune activation is the induction of ICD. ICD happens when tumor cells are subjected to exterior stimuli, mediating the physique’s antitumor immune response [47]. ICD is accompanied by the manufacturing of a wide range of damage-associated molecular patterns (DAMPs), such because the publicity of surface-exposed calreticulin (CALR) and the discharge of high-mobility group field 1 (HMGB1) [48,49,50,51]. Right here, we investigated the expression of CALR on the cell floor and the discharge of HMGB1 in numerous teams. The PBS, CR NPs, and PBS + laser remedies confirmed little cell floor CALR publicity (inexperienced), whereas the CR NPs + laser therapy resulted in enhanced publicity of CALR on the cell floor owing to CR-mediated PTT below laser irradiation (Fig. 3d). As well as, the CR NPs + laser therapy group confirmed much less HMGB1 staining within the cell nuclei in contrast with the opposite three teams, indicating a distinguished launch of HMGB1. Collectively, these outcomes indicated that CR NPs can broadly induce tumor cell apoptosis and ICD after laser irradiation.
In vivo NIR-II fluorescence imaging and biodistribution evaluation of CR NPs
Contemplating the superb in vitro properties of the CR NPs, we then studied the imaging properties of the NPs in vivo. DIR-loaded NPs (CR/DIR NPs) have been constructed to watch the in vivo biodistribution and accumulation of CR NPs utilizing a Colon26 tumor-bearing mouse mannequin. CR/DIR NPs have been ready utilizing the identical methodology as that used for the preparation of the CR NPs. When the tumor quantity reached a mean dimension of ≈ 100 mm3, CR/DIR NPs (50 µg ml− 1, 100 µL, primarily based on DIR) have been injected intravenously into the mice. The biodistribution was examined utilizing IVIS at totally different time factors after injection. Because the whole-body photos in Fig. 4a and b proven, fluorescent alerts have been primarily localized on tumor space and steadily enhanced over time, reaching a sign peak at 12 h after administration of CR/DIR NPs, which signifies glorious tumor accumulation of the NPs. Notably, the fluorescent alerts of tumors handled with CR/DIR NPs remained clear and robust even after 48 h, which offered a very long time window for PTT utility. Moreover, to guage the NPs distribution, ex vivo imaging of remoted important organs and tumors was carried out 48 h after the injection. As proven in Fig. 4c and d, vital fluorescence alerts have been nonetheless seen within the tumor and liver tissues 48 h after the injection of CR/DIR NPs, suggesting a powerful passive tumor-targeting capacity of CR/DIR NPs and liver clearance.
In vivo biodistribution evaluation and NIR-II FLI of CR NPs. (a) Imaging and (b) quantification evaluation of Colon26 tumor-bearing mice with CR/DIR NPs in numerous time factors (n = 3). (c and d) Ex vivo biodistribution and quantification of the dissected important organs and tumors. (e) NIR-II fluorescence photos and (f) the corresponding quantitative evaluation of CR NPs in 4T1 tumor-bearing mice at totally different occasions. 4T1 tumor-bearing mice have been intravenously injected with 100 µg of CR NPs (1.0 mg mL− 1) per mouse. Then, the real-time imaging was recorded and monitored at designated time intervals (0, 4, 8, 12, 24 and 36 h) post-injection (n = 3). NIR-II FLI situations: 808 nm, 1000 nm filter, 250 ms. White dashed circles point out tumor areas. (g) Ex vivo NIR-II FLI and (h) the corresponding quantification of main organs after 36 h administration of CR NPs
Inspired by these outcomes, we then evaluated the in vivo efficiency of CR NPs in NIR-II FLI utilizing a 4T1 tumor-bearing BALB/c mouse mannequin. First, the mice have been intravenously administered with CR NPs (100 µL, 1 mg mL− 1), and the NIR-II fluorescence photos have been subsequently recorded at totally different occasions. Within the Fig. 4e and f, the tumor profile was simply distinguishable, indicating that CR NPs might successfully accumulate on the tumor website. The NIR-II fluorescence intensities of the CR NPs at tumor areas progressively intensified inside 12 h and reached the plateau at 24 h after the injection, which was in accordance with the outcomes of NIR-I FLI (Fig. 4a and b). Subsequently, the optimum time level for PTT is 12 h after the injection. Moreover, the fluorescent alerts have been maintained within the tumor area for over 36 h, indicating a protracted therapeutic window for laser irradiation. Moreover, NIR-II ex vivo imaging of dissected tumors and important organs was carried out 36 h after injection to additional consider the biodistribution of the CR NPs. As proven in Fig. 4g and h, the CR NPs have been primarily retained within the liver, spleen, and tumor, suggesting good tumor-targeting capacity and potential metabolic organs. These imaging outcomes show that CR NPs possess favorable tumor-targeting capacity, which is conducive to tumor therapy.
Notably, these promising information show the potential for the applying of CR NPs in NIR-II imaging-guided surgical procedure. NIR-II FLI has emerged as a promising technique for exact image-guided tumor surgical procedure because of its deep tissue penetration and excessive spatial decision. The diminished background alerts, minimal tissue auto-fluorescence and low scattering within the NIR-II window allow clearer and extra exact imaging for tumor margins, which is essential for full tumor resection whereas minimizing harm to surrounding wholesome tissues. A number of research have proven the advantages of NIR-II FLI-guided most cancers surgical procedure, together with localizing cancers, evaluating surgical margins, guiding cytoreductive surgical procedure (CRS), tracing lymph nodes (LNs) and lymphatic vessels, and mapping particular anatomical constructions. These research have demonstrated the numerous prospects of NIR FLI guided surgical procedure in bettering surgical outcomes [52]. Moreover, the extended retention of CR NPs in tumor tissues supplies a considerable therapeutic window, permitting surgeons adequate time to carry out image-guided surgical procedure. General, CR NPs maintain vital potential to enhance the outcomes of surgical interventions by excellent tumor focusing on and imaging capabilities.
CR NPs-mediated in vivo photothermal remedy
Based mostly on the environment friendly in vitro PTT impact and glorious tumor accumulation of CR NPs, we then studied the PTT exercise of the CR NPs within the Colon26 tumor mannequin. When tumor dimension reached 100 mm3, the mice have been randomly divided into 4 teams (5 mice per group): (1) PBS, (2) PBS + laser, (3) CR NPs, and (4) CR NPs + laser. First, the mice have been intravenously injected with PBS and CR NPs; the tumors of mice in teams 2 and 4 have been then constantly irradiated for 10 min 12 h post-injection in accordance with the in vivo imaging information. The true-time photothermal photos and temperatures of the mice within the laser-treated teams have been recorded with an IR thermal digital camera to verify the photothermal results of the CR NPs. Determine 5a and b illustrated that the tumor area’s temperature within the CR NPs + laser therapy group quickly elevated to 52 °C inside 2 min of irradiation and retained this temperature throughout the remainder interval of irradiation. The management group (PBS + laser) confirmed somewhat improve in temperature (ΔT ≈ 4 °C) below the identical therapy situations, demonstrating that the photothermal impact induced by the laser alone was negligible and didn’t have a PTT impact in vivo.
In vivo CR NPs-mediated photothermal remedy. (a) Infrared thermal photos and (b) corresponding temperature profiles on the tumor websites of Colon26 tumor-bearing mice handled with PBS or CR NPs below irradiation (808 nm, 1.0 W cm− 2) at totally different time factors (n = 5). (c) Tumor development profiles of 4 teams through the therapy course. Statistical significance was calculated through two-way ANOVA. (d-e) The tumor pictures (d) and (e) weights of dissected tumors of mice in every group on the 14th day. Statistical significance was calculated through one-way ANOVA. (f) Physique weight of mice among the many therapy teams. CR NPs injection and laser irradiation got solely as soon as
To additional consider the in vivo antitumor impact of CR NPs below laser irradiation, the tumor sizes and physique weights have been monitored each different day after remedy. Two days after PTT, black scars have been noticed within the tumor space from the CR NPs + laser group, whereas insignificant modifications have been discovered within the mice from the PBS + laser group. In Fig. 5c and Determine S7, the tumors of teams (1), (2) and (3) grew constantly with comparable excessive development charges, and the typical tumor quantity elevated to roughly 1500 mm3 inside 14 days. This means that CR NPs or laser irradiation alone haven’t any therapeutic impact. Notably, the NPs + laser therapy resulted in full tumor eradication. These outcomes point out a big in vivo photothermal therapeutic impact of CR NPs, which aligned with the in vitro findings. On the 14th day after therapy, the mice in all teams have been sacrificed and the tumors have been photographed and weighed (Fig. 5d and e). The tumor weight was in good settlement with the tendency of tumor development profiles, additional verifying the superb in vivo photothermal ablation impact of CR NPs. The physique weights of the mice have been comparable, with a slight improve all through the therapy interval, demonstrating passable biocompatibility and restricted negative effects of CR NPs in vivo for PTT (Fig. 5f). Notably, a single-dose administration of CR NPs and as soon as irradiation have been carried out, which resulted in full tumor elimination, additional demonstrating the superior PTT efficacy of CR NPs for in vivo remedy.
To additional consider the biosafety of CR NPs, histological staining of main organs and blood biochemical evaluation of the handled mice have been carried out on the finish of therapy. As proven in Determine S8, the histological evaluation of the most important organs of all mice confirmed no vital tissue harm or irritation after therapy, additional confirming the superb in vivo biosafety of the CR NPs. The physiological security of the CR NPs was estimated utilizing routine blood and biochemistry evaluation (Determine S9). Varied blood parameters confirmed no vital variations among the many 4 teams after therapy, indicating that the CR NPs didn’t present systemic toxicity or dangerous impacts on livers and kidneys of the mice. Taken collectively, CR NPs are a promising candidate for exact NIR-II FLI-guided PTT in vivo, with glorious theranostic functionality and negligible negative effects.
Antitumor immune reminiscence evaluation
Given the entire tumor eradication within the Colon26 tumor mannequin after therapy with CR NPs + laser irradiation, we puzzled whether or not antitumor reminiscence could possibly be induced by CR NPs. After therapy, tumor-free mice in one other batch of mice have been re-challenged with 2.0 × 106 Colon26 tumor cells on day 52 after the preliminary therapy (Fig. 6a). Age-matched mice injected with Colon26 cells (2 × 106) cells have been used as controls. The volumes of the tumors have been measured each alternate day. Consequently, the 4/5 of mice did not kind tumors within the re-challenged mice receiving the therapy of CR NPs + laser irradiation, whereas all six mice within the management group developed quickly rising tumors (Fig. 6b), indicating that antitumor immune reminiscence is developed after publicity to CR NPs. To elucidate the basic mechanism, stream cytometry was used to quantify the memory-associated T cells within the spleen. The outcomes of Fig. 6c and Determine S10 indicated that CD3+, CD3+CD8+, and CD3+CD4+ T lymphocytes within the spleens of rechallenge-resistant mice have been notably elevated compared with the management group. The stream cytometric evaluation additional revealed that effector reminiscence T cells (Tem, CD62L+CD44−) have been noticeably elevated each in CD4+ and CD8+ T cells, and central reminiscence T cells (Tcm, CD62L+CD44+) in CD4+ T cells have been additionally considerably elevated (Fig. 6d–f). These outcomes clearly point out that the CR NPs + laser therapy can successfully inhibit tumor development and promote the formation of long-term antitumor reminiscence, thereby probably stopping tumor metastasis.
Antitumor immune reminiscence evaluation. (a) Mice cured by CR NPs + laser therapy have been re-challenged by Colon26 cells (2 × 106 cells) on day 52 after the preliminary therapy. (b) Tumor quantity of every re-challenged mouse. (c) Quantification of CD3+, CD3+CD8+, CD3+CD4+ T lymphocytes within the spleen. (d-f) Move cytometric (FCM) evaluation and quantitative information of central reminiscence T (Tcm, CD62L+CD44+) cells and effector reminiscence T (Tem, CD62L−CD44+) cells subset from CD8+ and CD4+ T cells within the spleen. Knowledge are proven because the imply ± SD, n = 5–6. Statistical significance was calculated through a two-tailed Pupil’s t-test
Anti-primary and metastatic tumor efficacy of CR NPs in 4T1 breast most cancers mannequin
Given the induction of long-term antitumor immune reminiscence in Colon26 tumor-bearing mice, we additional investigated whether or not CR NPs + laser irradiation might exert concurrently anti-primary and anti-metastatic tumor results utilizing an orthotopic 4T1 murine mammary carcinoma mannequin which might induce lung metastasis throughout main tumor improvement. We first assessed the anti-primary tumor efficiency on this refractory tumor mannequin. When the tumor volumes reached 100 mm3, the mice have been handled with PBS or CR NPs + laser irradiation 12 h after injection. The tumor volumes and physique weights have been recorded each different day through the therapy interval. The outcomes confirmed that CR NPs + laser irradiation successfully inhibited orthotopic 4T1 tumor development, though no tumor eradication was noticed owing to its resistance to remedy (Fig. 7a–c), demonstrating the large applicability of CR NPs-mediated PTT in numerous tumor varieties. Equally, CR + laser therapy didn’t lead to any vital alterations in animal physique weight in comparison with PBS therapy (Fig. 7d). The survival time of the mice within the CR NPs + laser therapy group was additionally drastically extended (Fig. 7e), whereas tumors in mice handled with PBS alone grew quickly, with a mean lifespan of solely 20–26 days. These outcomes counsel that CR NPs + laser irradiation is extremely environment friendly and protected for the in vivo photothermal ablation of extremely metastatic tumors.
As most cancers metastasis is the principle reason for tumor-associated dying, we subsequently investigated whether or not CR NPs + laser irradiation might cut back lung metastasis of 4T1 tumors after therapy in one other batch of tumor-bearing mice. Tumor development of mice within the CRs + laser group was drastically inhibited, which was in step with the outcomes of the primary batch of mice (Determine S11). The tumors and lungs of the mice have been collected on day 30 after therapy to guage the antimetastatic results. As displayed in Fig. 7f and g, the first tumor weight was considerably decrease in CR NPs + laser-treated mice than in management mice, and the variety of pulmonary metastatic tumor nodules on the lung floor was additionally remarkably diminished in handled mice compared with the management group. The lungs have been sectioned for histological analysis, and nearly no metastatic lesions have been noticed within the CR NPs + laser-treated group (Fig. 7h), whereas vital metastatic foci have been found within the management group’s lungs, indicating that CR NPs-based PTT successfully inhibits the lung metastasis of tumor cells. Subsequently, the CR NPs-based PTT can induce a potent systemic immune response and forestall tumor metastasis.
Anti-primary and metastatic tumor efficacy on a 4T1 breast most cancers mannequin through the use of CR NPs. (a) Tumor quantity development curves from the 2 handled teams (n = 5). Statistical significance was calculated through two-way ANOVA. Corresponding tumor development curves in (b) PBS and (c) CR NPs + laser teams. (d) Physique weights of the handled mice. (e) Survival curves (n = 5). Statistical significance was calculated through Log-rank (Mantel-Cox) check. (f) Tumor weights and (g) the variety of metastatic nodules on lung floor on the endpoint. Statistical significance was calculated through unpaired Pupil’s-test. (h) Typical hematoxylin and eosin (H&E) staining of lungs from the 2 teams
