Synthesis and characterization of the CR NPs
First, the D–π–A–π–D structured CR-TPE-T was synthesized via a single-step condensation between croconic acid and N, N-bis(4-(1,2,2-triphenylvinyl) phenyl) thiophen-2-amine in 1:2 equal based mostly on the earlier experiences [32] (Scheme S1). The molecular geometry and digital distribution of CR-TPE-T have been studied by density useful idea (DFT) calculations. As proven in Fig. 1a, the optimized ground-state (S0) geometry of CR-TPE-T confirmed an axisymmetric configuration in area; 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 very best occupied molecular orbital (HOMO) have been properly 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 inside the molecule. The HOMO and LUMO vitality 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 idea (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 answer. (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 in the dead of night at room temperature
For in vivo functions, the hydrophobic CR-TPE-T was then fabricated into nanoparticles (NPs) (CR NPs) via nanoprecipitation utilizing DSPE-mPEG2000 because the encapsulation matrix. In accordance with 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 lengthening over 1000 nm, matching very properly 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 masking 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 ends in 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 via the EPR impact. Moreover, the CR NPs zeta potential was − 33.7 ± 1.71 mV, which favors good stability in a physiological surroundings (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 answer after storage at room temperature for five weeks, implying good colloidal stability of the CR NPs (Fig. 1f). These outcomes reveal 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 photographs (Fig. 2a) recorded by a thermal digicam, the CR NPs exhibited an obvious photothermal impact on 808-nm laser irradiation. The laser energy density results on the temperature adjustments 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 constructive 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 adjustments (Fig. 2c and S4a). These outcomes point out controllable photothermal habits. Photothermal stability is a vital 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 very 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 perform via photothermal results with out concurrent photodynamic exercise.
In vitro NIR-II FLI and PTT properties of CR NPs. (a) Infrared (IR) thermal photographs of 200 µg mL–1 CR NPs aqueous answer 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 answer with different laser energy densities (200 µg mL–1, 808 nm). (d) Photothermic stability of 200 µg mL− 1 CR NPs in aqueous answer (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 answer of CR NPs at completely 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 regular 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 wonderful biocompatibility of CR NPs for biomedical and medical functions. In distinction, important photothermal cytotoxicity was noticed after remedy 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. Comparable outcomes have been additionally obtained in 4T1 cells, exhibiting that CR NPs + laser irradiation might considerably suppress the cell viability in a concentration-dependent method (Determine S6a). In the meantime, we observed that 4T1 cells seemed to be extra delicate to CR NPs-induced photothermal impact than Colon26 since decrease concentrations of CR NPs corresponding to 2.5 and 5 µg ml− 1 have urged outstanding inhibition to viability upon irradiation. To additional examine the photothermal impact of CR NPs, we then performed dwell (inexperienced fluorescence)/useless (purple 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. Pink fluorescence was clearly noticed within the CR NPs + laser irradiation group, and nearly 70% of cells underwent demise after irradiation. This illustrates the wonderful PTT capability of CR NPs towards Colon26 cells, which is in step with CCK-8 findings. Moreover, cell apoptosis was studied by movement 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 movement cytometry after completely 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 quite a lot 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 remedy 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 remedy group confirmed much less HMGB1 staining within the cell nuclei in contrast with the opposite three teams, indicating a outstanding 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 wonderful 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 observe 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, based mostly on DIR) have been injected intravenously into the mice. The biodistribution was examined utilizing IVIS at completely different time factors after injection. Because the whole-body photographs in Fig. 4a and b proven, fluorescent alerts have been primarily localized on tumor space and regularly enhanced over time, reaching a sign peak at 12 h after administration of CR/DIR NPs, which signifies wonderful 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 supplied a very long time window for PTT software. Moreover, to guage the NPs distribution, ex vivo imaging of remoted important organs and tumors was performed 48 h after the injection. As proven in Fig. 4c and d, important 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 skill 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 photographs and (f) the corresponding quantitative evaluation of CR NPs in 4T1 tumor-bearing mice at completely different instances. 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 photographs have been subsequently recorded at completely different instances. Within the Fig. 4e and f, the tumor profile was simply distinguishable, indicating that CR NPs might successfully accumulate on the tumor web site. 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 very important organs was performed 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 skill and attainable metabolic organs. These imaging outcomes reveal that CR NPs possess favorable tumor-targeting skill, which is conducive to tumor remedy.
Notably, these promising information reveal the potential for the appliance 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 resulting from its deep tissue penetration and excessive spatial decision. The decreased 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 ample time to carry out image-guided surgical procedure. Total, CR NPs maintain important potential to enhance the outcomes of surgical interventions via 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 wonderful 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 keeping with the in vivo imaging information. The true-time photothermal photographs and temperatures of the mice within the laser-treated teams have been recorded with an IR thermal digicam 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 remedy group quickly elevated to 52 °C inside 2 min of irradiation and retained this temperature throughout the remaining interval of irradiation. The management group (PBS + laser) confirmed just a little enhance in temperature (ΔT ≈ 4 °C) below the identical remedy 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 photographs 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 completely different time factors (n = 5). (c) Tumor development profiles of 4 teams throughout the remedy course. Statistical significance was calculated through two-way ANOVA. (d-e) The tumor images (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 remedy 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 adjustments 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 remedy resulted in full tumor eradication. These outcomes point out a major in vivo photothermal therapeutic impact of CR NPs, which aligned with the in vitro findings. On the 14th day after remedy, 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 wonderful in vivo photothermal ablation impact of CR NPs. The physique weights of the mice have been comparable, with a slight enhance all through the remedy interval, demonstrating passable biocompatibility and restricted unintended 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 performed, 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 performed on the finish of remedy. As proven in Determine S8, the histological evaluation of the most important organs of all mice confirmed no important tissue harm or irritation after remedy, additional confirming the wonderful in vivo biosafety of the CR NPs. The physiological security of the CR NPs was estimated utilizing routine blood and biochemistry evaluation (Determine S9). Numerous blood parameters confirmed no important variations among the many 4 teams after remedy, 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 wonderful theranostic functionality and negligible unintended effects.
Antitumor immune reminiscence evaluation
Given the whole tumor eradication within the Colon26 tumor mannequin after remedy with CR NPs + laser irradiation, we questioned whether or not antitumor reminiscence could possibly be induced by CR NPs. After remedy, 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 remedy (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. In consequence, the 4/5 of mice did not kind tumors within the re-challenged mice receiving the remedy 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, movement 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 movement 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 remedy 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 remedy have been re-challenged by Colon26 cells (2 × 106 cells) on day 52 after the preliminary remedy. (b) Tumor quantity of every re-challenged mouse. (c) Quantification of CD3+, CD3+CD8+, CD3+CD4+ T lymphocytes within the spleen. (d-f) Movement 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. Information are proven because the imply ± SD, n = 5–6. Statistical significance was calculated through a two-tailed Scholar’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 major tumor growth. 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 throughout the remedy 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 huge applicability of CR NPs-mediated PTT in numerous tumor varieties. Equally, CR + laser remedy didn’t lead to any important alterations in animal physique weight in comparison with PBS remedy (Fig. 7d). The survival time of the mice within the CR NPs + laser remedy group was additionally vastly 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 primary reason for tumor-associated demise, we subsequently investigated whether or not CR NPs + laser irradiation might cut back lung metastasis of 4T1 tumors after remedy in one other batch of tumor-bearing mice. Tumor development of mice within the CRs + laser group was vastly inhibited, which was in line 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 remedy 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 decreased 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 important 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 Scholar’s-test. (h) Typical hematoxylin and eosin (H&E) staining of lungs from the 2 teams
