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In this study, videothermometry's application in detecting mammary tumors in dogs is explored in-depth. The research hypothesizes that this technique can effectively identify cancerous tissues during surgery by analyzing thermal patterns. The methodology involved comparing thermal imaging results from dogs with palpable mammary nodules against a control group, focusing on capturing real-time thermal patterns. Results were significant, showing distinct thermal patterns in carcinomas. This indicates videothermometry's capability in accurately identifying micro metastases and differentiating between neoplastic and non-neoplastic changes. The study concludes that videothermometry has considerable potential in enhancing surgical precision, especially in tumor resection and safety margin definition, but emphasizes the need for further research to thoroughly understand the thermal signatures of various mammary tumors in dogs.

Background: Canine mammary tumours (CMTs) are the most common neoplasm appearing in female dogs and are considered the equivalent animal model of human breast cancer. However, in the literature, there is a gap for ultrasonic characterisation of these tumours. In this study, experimental measurements for acoustic attenuation and propagation speed of three surgically excised malignant CMTs were implemented., Methods: The three tumours were fixed in formaldehyde for up to 72 h and a total of five sample pieces were sectioned from the three tumours to account for the varied morphology observed along the tumours. The through-transmission and pulse-echo techniques were employed for experimental measurements of the acoustic attenuation and propagation speed., Results: Acoustic propagation speed of the five samples as measured at 2.7 MHz was in the range of 1568-1636 m/s. Correspondingly, acoustic attenuation was in the range of 1.95-3.45 dB/cm.MHz. Variations in both speed and attenuation were observed between samples acquired from the same tumour., Conclusions: Present findings suggest that both acoustic attenuation and propagation speed of CMTs are higher than normal canine tissues due to increased heterogeneity and varied morphology visually observed between the tumour specimens and evidenced by histological examination. Nevertheless, experimental results could aid in enhancing the use of ultrasound in the diagnosis and treatment of CMTs as well as provide essential data for comparative oncology., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Since gamma-glutamyl transpeptidase (GGT) is highly and locally expressed in human breast cancer, a GGT-enzymatically activatable fluorescent probe, gamma-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG), has been developed to detect the human breast cancer area with high performance. In this study, GGT expression and the efficacy of gGlu-HMRG on visualization were investigated in canine mammary gland tumors (MGT). Seventeen non-fixed fresh-frozen MGT specimens and each peritumoral control tissue were utilized. The GGT mRNA levels were highly observed in the tumor specimens compared with the control. GGT immunostaining was mostly observed on the cell membrane and cytosol of the alveolar and duct mammary epithelium of MGT tissues. These signals were strongly positive in several cases while they were mild to not observed in other cases. When gGlu-HMRG solution was dropped to the non-fixed tissue pieces of MGT or control tissues, the fluorescence intensities (FIs) were measured using Maestro in-vivo imaging device. FIs in MGT tissues were significantly higher than each control tissue 20 min after treatment. Based on Youden index method, the maximum sensitivity and specificity of FI was 82.4% and 82.4%. These findings suggest that GGT is highly expressed in several MGTs in dogs and gGlu-HMRG could visualize at least a part of MGT tissues in dogs. Nevertheless, it should be needed to assess the false-negative areas more carefully in canine than human cases.

Optical coherence tomography (OCT) uses near-infrared light waves to generate real-time, high-resolution images on the microscopic scale similar to low power histopathology. Previous studies have demonstrated the use of OCT for real-time surgical margin assessment for human breast cancer. The use of OCT for canine mammary tumours (CMT) could allow intra-operative visualisation of residual tumour at the surgical margins. The purpose of this study was to assess OCT imaging for the detection of incomplete tumour resection following CMT surgery. We hypothesized that the OCT images would have comparable features to histopathological images of tissues at the surgical margins of CMT resections along with a high sensitivity of OCT detection of incomplete surgical excision of CMT. Thirty surgical specimens were obtained from nineteen client-owned dogs undergoing surgical resection of CMT. OCT image appearance and characteristics of adipose tissue, skin, mammary tissue and mammary tumour at the surgical margins were distinct and different. The OCT images of normal and abnormal tissues at the surgical margins were utilized to develop a dataset of OCT images for observer evaluation. The sensitivity and specificity for ex vivo images were 83.3% and 82.0% (observer 1) and 70.0% and 67.9% (observer 2). The sensitivity and specificity for in vivo images were 70.0% and 89.3% (observer 1) and 76.7% and 67.9% (observer 2). These results indicate a potential use of OCT for surgical margin assessment for CMT to optimize surgical intervention and clinical outcomes. Improved training and experience of observers may improve sensitivity and specificity., (© 2020 John Wiley & Sons Ltd.)

Background: Thoracic radiography in awake cats is a common procedure for the evaluation of pulmonary metastasis in feline mammary gland carcinoma (MGC). However, due to poor sensitivity, computed tomography (CT) is progressively taking its place. To perform CT in animals, general anesthesia is normally preferred but can cause lung atelectasis, affecting lung interpretation. Besides, MGC is often found in senile cats that are concurrently affected with other diseases, increasing anesthetic risk. Therefore, this study was aimed at comparing the effect of anesthesia on lung atelectasis observed through CT in clinically healthy cats and comparing the feasibility of non-anesthetic CT with non-anesthetic radiography in the detection of lung lesions in feline MGC. Thoracic CTs from anesthetized, clinically healthy cats and non-anesthetized either clinically healthy cats or MGC-affected cats were reviewed. In clinically healthy cats, motion artifacts and characteristics of lung atelectasis were observed and compared. In MGC-affected cats, motion artifacts were observed and compared to clinically healthy cats, and the number of MGC-affected cats, the number and characteristics of lung lesions were compared between non-anesthetic thoracic CT and radiography., Results: Anesthesia significantly increased lung CT attenuation (P = 0.0047) and was significantly correlated with lung atelectasis (OR = 15; CI 2.02-111.18; P = 0.0081), particularly of the cranial lung lobe. Nonetheless, significantly higher motion artifacts in the caudal thoracic area were found in non-anesthetized healthy cats (P = 0.0146), but comparable low motion artifacts were observed in anesthetized healthy and MGC-affected cats. Non-anesthetic CT revealed higher numbers of MGC-affected cats and pulmonary nodules with a significantly lower nodular diameter (P = 0.0041) than those observed on radiographs. The smallest nodular diameters detected on radiographs and CT were 2.5 and 1.0 mm, respectively. Furthermore, CT showed additional information such as intra-thoracic lymphadenopathy, that could not be seen on radiographs., Conclusions: Despite the motion artifacts, CT without anesthesia is a sensitive technique as it provides better lung inflation. Furthermore, compared to non-anesthetic radiography, non-anesthetic CT provided more information such as higher number of pulmonary nodules of a smaller size, including more distinct intra-thoracic lesions, such as lymphadenopathy, in MGC-affected cats.

Purpose: To assess the feasibility of using diffusion-time-dependent diffusional kurtosis imaging (tDKI) to measure cellular-interstitial water exchange time (τ ex ) in tumors, both in animals and in humans., Methods: Preclinical tDKI studies at 7 T were performed with the GL261 glioma model and the 4T1 mammary tumor model injected into the mouse brain. Clinical studies were performed at 3 T with women who had biopsy-proven invasive ductal carcinoma. tDKI measurement was conducted using a diffusion-weighted STEAM pulse sequence with multiple diffusion times (20-800 ms) at a fixed echo time, while keeping the b-values the same (0-3000 s/mm 2 ) by adjusting the diffusion gradient strength. The tDKI data at each diffusion time t were used for a weighted linear least-squares fit method to estimate the diffusion-time-dependent diffusivity, D(t), and diffusional kurtosis, K(t)., Results: Both preclinical and clinical studies showed that, when diffusion time t ≥ 200 ms, D(t) did not have a noticeable change while K(t) decreased monotonically with increasing diffusion time in tumors and t ≥ 100 ms for the cortical ribbon of the mouse brain. The estimated τ ex averaged median and interquartile range (IQR) of GL261 and 4T1 tumors were 93 (IQR = 89) ms and 68 (78) ms, respectively. For the cortical ribbon, the estimated τ ex averaged median and IQR were 41 (34) ms for C57BL/6 and 30 (17) ms for BALB/c. For invasive ductal carcinoma, the estimated τ ex median and IQR of the two breast cancers were 70 (94) and 106 (92) ms., Conclusion: The results of this proof-of-concept study substantiate the feasibility of using tDKI to measure cellular-interstitial water exchange time without using an exogenous contrast agent., (© 2021 John Wiley & Sons, Ltd.)

Precise diagnosis and prognosis are key in prevention and reduction of morbidity and mortality in all types of cancers. Here we show that changes in the collagen fibres in the main histological subtypes of canine mammary gland carcinomas are directly associated with the tumour behaviour and the animal survival time and could become a useful tool in helping with diagnosis. Imaging by second harmonic generation and multiphoton excited fluorescence microscopy were performed to evaluate the collagen and cellular segment parameters in cancer biopsies. We present a retrospective study of 45 cases of canine mammary cancer analysing 836 biopsies regions including normal mammary gland tissue, benign mixed tumours, carcinoma in mixed tumour, carcinosarcoma, micropapillary carcinoma and solid carcinoma. The image analyses and the comparison between the tumour types allowed to assess the collagen fibre changes during tumour progression. We demonstrate that the collagen parameters correlate with the clinical and pathological data, the results show that in neoplastic tissues, the collagen fibres are more aligned and shorter as compared to the normal tissues. There is a clear association of the mean fibre length with the dogs survival times, the carcinomas presenting shorter collagen fibres indicate a worse survival rate.

Development of imaging methods capable of furnishing tumor-specific morphological, functional, and molecular information is paramount for early diagnosis, staging, and treatment of breast cancer. Ultrasound (US) and optoacoustic (OA) imaging methods exhibit excellent traits for tumor imaging in terms of fast imaging speed, ease of use, excellent contrast, and lack of ionizing radiation. Here, we demonstrate simultaneous tomographic whole body imaging of optical absorption, US reflectivity, and speed of sound (SoS) in living mice. In vivo studies of 4T1 breast cancer xenografts models revealed synergistic and complementary value of the hybrid imaging approach for characterizing mammary tumors. While neovasculature surrounding the tumor areas were observed based on the vascular anatomy contrast provided by the OA data, the tumor boundaries could be discerned by segmenting hypoechoic structures in pulse-echo US images. Tumor delineation was further facilitated by enhancing the contrast and spatial resolution of the SoS maps with a full-wave inversion method. The malignant lesions could thus be distinguished from other hypoechoic regions based on the average SoS values. The reported findings corroborate the strong potential of the hybrid imaging approach for advancing cancer research in small animal models and fostering development of new clinical diagnostic approaches., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: To investigate the feasibility of using diffusion MRI (dMRI) and dynamic contrast-enhanced (DCE) MRI to evaluate the treatment response of metronomic chemotherapy (MCT) in the 4T1 mammary tumor model of locally advanced breast cancer., Methods: Twelve Balb/c mice with metastatic breast cancer were divided into treated and untreated (control) groups. The treated group (n = 6) received five treatments of anti-metabolite agent 5-Fluorouracil (5FU) in the span of two weeks. dMRI and DCE-MRI were acquired for both treated and control groups before and after MCT. Immunohistochemically staining and measurements were performed after the post-MRI measurements for comparison., Results: The control mice had significantly (p<0.005) larger tumors than the MCT treated mice. The DCE-MRI analysis showed a decrease in contrast enhancement for the control group, whereas the MCT mice had a more stable enhancement between the pre-chemo and post-chemo time points. This confirms the antiangiogenic effects of 5FU treatment. Comparing amplitude of enhancement revealed a significantly (p<0.05) higher enhancement in the MCT tumors than in the controls. Moreover, the MCT uptake rate was significantly (p<0.001) slower than the controls. dMRI analysis showed the MCT ADC values were significantly larger than the control group at the post-scan time point., Conclusion: dMRI and DCE-MRI can be used as potential biomarkers for assessing the treatment response of MCT. The MRI and pathology observations suggested that in addition to the cytotoxic effect of cell kills, the MCT with a cytotoxic drug, 5FU, induced changes in the tumor vasculature similar to the anti-angiogenic effect., Competing Interests: The authors have declared that no competing interests exist.

Breast cancer is the second most commonly diagnosed malignancy among women globally. Past MRI studies have linked a high animal fat diet (HAFD) to increased mammary cancer risk in the SV40Tag mouse model of triple-negative breast cancer. Here, serial MRI examines tumor progression and measures the arterial blood volume feeding mammary glands in low fat diet (LFD) or HAFD fed mice. Virgin female C3(1)SV40Tag mice (n = 8), weaned at 3 weeks old, were assigned to an LFD (n = 4, 3.7 kcal/g, 17.2% kcal from vegetable oil) or an HAFD (n = 4, 5.3 kcal/g, 60% kcal from lard) group. From ages 8 to 12 weeks, weekly fast spin echo MR images and time-of-flight (TOF) MR angiography of inguinal mammary glands were acquired at 9.4 T. Following in vivo MRI, mice were sacrificed. Inguinal mammary glands were excised and fixed for ex vivo MRI and histology. Tumor, blood, and mammary gland volumes for each time point were measured from manually traced regions of interest; tumors were classified as invasive by histopathology-blinded observers. Our analysis confirmed a strong correlation between total tumor volume and blood volume in the mammary gland. Tumor growth rates from weeks 8-12 were twice as high in HAFD-fed mice (0.42 ± 0.14/week) as in LFD-fed mice (0.21 ± 0.03/week), p < 0.004. Mammary gland blood volume growth rate was 2.2 times higher in HAFD mice (0.29 ± 0.11/week) compared with LFD mice (0.13 ± 0.06/week), p < 0.02. The mammary gland growth rate of HAFD-fed mice (0.071 ± 0.011/week) was 2.7 times larger than that of LFD-fed mice (0.026 ± 0.009/week), p < 0.01. This is the first non-invasive, in vivo MRI study to demonstrate a strong correlation between an HAFD and increased cancer burden and blood volume in mammary cancer without using contrast agents, strengthening the evidence supporting the adverse effects of an HAFD on mammary cancer. These results support the potential future use of TOF angiography to evaluate vasculature of suspicious lesions., (© 2020 John Wiley & Sons, Ltd.)

Purpose: Negative surgical margins (NSMs) have favorable prognostic implications in breast tumor resection surgery. Fluorescence image-guided surgery (FIGS) has the ability to delineate surgical margins in real time, potentially improving the completeness of tumor resection. We have recently developed indocyanine green (ICG)-loaded self-assembled hyaluronic acid (HA) nanoparticles (NanoICG) for solid tumor imaging, which were shown to enhance intraoperative contrast., Procedures: This study sought to assess the efficacy of NanoICG on completeness of breast tumor resection and post-surgical survival. BALB/c mice bearing iRFP + /luciferase + 4T1 syngeneic breast tumors were administered NanoICG or ICG, underwent FIGS, and were compared to bright light surgery (BLS) and sham controls., Results: NanoICG increased the number of complete resections and improved tumor-free survival. This was a product of improved intraoperative contrast enhancement and the identification of a greater number of small, occult lesions than ICG and BLS. Additionally, NanoICG identified chest wall invasion and predicted recurrence in a model of late-stage breast cancer., Conclusions: NanoICG is an efficacious intraoperative contrast agent and could potentially improve surgical outcomes in breast cancer.

Introduction: Current methods of intraoperative margin assessment in breast conserving surgery are impractical, unreliable, or time consuming. We hypothesized that intraoperative near-infrared (NIR) imaging with an FDA-approved NIR optical contrast agent could identify canine mammary tumors, a spontaneous large animal model of human breast cancer, during surgery., Methods: Dogs with mammary tumors underwent a standard of care lumpectomy or mastectomy with wide surgical margins 20 hours after indocyanine green administration (3 mg/kg IV). During surgery, NIR imaging was performed on tumors and wound margins in situ and tumors and lymph nodes ex vivo. Following resection, the wound bed was examined for residual fluorescence. Fluorescence intensity was determined by signal-to-background ratio (SBR). All tumors, areas of residual fluorescence, and lymph nodes underwent histopathologic analysis., Results: There were 41 mammary tumors in 16 female dogs. Twenty tumors were malignant and 21 were benign. Twenty-eight tumors were fluorescent (mean SBR 1.5±0.2). Sensitivity of fluorescence for all malignant tumors was 80% (16/20) and 93.3% (14/15) for malignant tumors > 2 cm. Specificity for malignancy was low (< 2cm = 55%; > 2cm = 30%). Tumors > 2 cm were more likely to be fluorescent (OR 6.05, 95% CI 1.50-24.44, P = 0.011) but not more likely to be malignant (OR 3.09, 95% CI 0.86-11.14, P = 0.085) than tumors ≤ 2 cm. Four out of seven inguinal lymph nodes excised in the mastectomy specimen fluoresced. All four drained malignant tumors; however only 2/4 contained metastatic disease., Conclusion: Systemic ICG accumulates reliably in malignant canine mammary tumors > 2 cm. Although no tumor margins fluoresced, a wider margin of normal tissue is removed in canine mastectomy, making direct comparisons with breast conserving surgery difficult. Targeted NIR imaging agents are likely required to improve detection of smaller tumors and improve the specificity of NIR imaging for residual disease and metastatic lymph node detection., Competing Interests: The authors have declared that no competing interests exist.

Purpose: Tumor development and metastasis are dependent on tumor infiltrating immune cells which form a characteristic tumor microenvironment (TME). Activated monocytes secrete the protein heterodimer S100A8/A9 promoting TME formation. Monocyte-dependent proteases facilitate local tumor cell invasion by degradation of the extracellular matrix. We aimed for target specific in vivo imaging of S100A8 and proteases to provide differentiating biomarkers for local tumor growth and metastatic potential., Procedures: Murine breast cancer cells of the 4T1 model with graduated metastatic potential (4T1 and 4T07: both hematogenous metastasis > 168FAR: lymph-node metastasis > 67NR: no metastasis) were orthotopically implanted into female BALB/c mice. At 4 mm size, tumors were investigated by injecting the protease-specific probe ProSense 750EX (PerkinElmer, 4T1 n = 7, 4T07 n = 10, 168FAR n = 16, 67NR n = 15) and anti-S100A8-Cy5.5 (n = 6 each) and performing fluorescence reflectance imaging at 0 and 24 h after injection. In vivo imaging was validated with immunohistochemistry., Results: At 24 h, S100A8-specific signals in 4T1 and 4T07 were significantly higher (1714.05/1683.45 AU) as compared to 168FAR and 67NR (174.85/167.95 AU, p = 0.0012/p = 0.0003), reflecting the capability of hematogenous spread. Protease-specific signals were significantly higher in 4T1 and 4T07 (348.01/409.93 AU) as compared to 168FAR (214.91 AU) and 67NR (129.78 AU p < 0.0001 each), reflecting local vessel invasion and tumor cell shedding. Immunohistology supported the in vivo imaging results., Conclusions: Non-invasive in vivo imaging of S100A8 and monocytic proteases allows for differentiation of the tumors' local invasive and systemic metastatic potential in reflecting the TME formation. While proteases augment local tumor cell invasion, solid metastases seem to be dependent on a pro-tumoral microenvironment.

Black titanium dioxide (TiO2) nanoparticles have attracted great attention due to their application in photothermal therapy (PTT). However, single-mode phototherapy has the risk of recurrence, and the high-dose laser usually imposed to improve the PTT performance can bring a potential threat to security. Here, polydopamine (PDA)-coated black TiO2 (b-P25@PDA) nanoparticles with a core-shell structure were synthesized for enhanced PTT; then, synergistic phototherapy nanoprobes (b-P25@PDA-Ce6 (Mn)) were constructed by coupling chlorin e6 (Ce6) and chelating Mn2+ for simultaneous photodynamic therapy (PDT)/PTT and magnetic resonance (MR) imaging, in which a low-dose laser was used and imaging-guided phototherapy with high efficiency and high safety was achieved. The prepared nanoprobes showed high photothermal conversion efficiency (32.12%), high reactive oxygen generation and excellent MR imaging. In the 4T1 tumor-bearing nude mouse model, the tumors completely disappeared under the combination of PDT/PTT with a low-dose laser but were only partially inhibited by single PDT and single PTT. The current work developed a multifunctional black TiO2-based nanoprobe for enhanced synergistic PDT/PTT and MR imaging, which will be important for the safe and efficient visualized theranostics of cancers.

The assessment of vascular permeability of malignant tumor plays an important role in the diagnosis and treatment of cancer. Dynamic contrast-enhanced magnetic resonance image (DCE-MRI) using Gd-encapsulated carbonaceous dots and Gd-DTPA-BMA as contrast agents was performed in 4T1 mouse breast cancer and HCC827 human non-small-cell lung cancer (NSNLC) xenograft models. Histopathological parameters of tumor vascularity microvessel density (MVD), microvessel area (MVA), endothelial area (EA) and α-SMA CD31 Co-expression (α-SMA/CD31%) were compared with the DCE-MRI parameters. Results demonstrated that DCE-MRI with the new nanoparticle Gd@C-dots can noninvasively evaluate vascular permeability. K trans measured by DCE-MRI with Gd@C-dots is an accurate parameter for the characterization of tumor permeability. EA is a reliable microvessel parameter to evaluate vessel permeability., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Triple negative breast cancer (TNBC), the most aggressive breast cancer type, is associated with high mortality and recurrence rates. An active-targeted strategy based on homing peptides is an effective approach to diagnose and treat cancer as it can deliver imaging agents or therapeutic drugs into desired tissues and accumulate less into off-target tissues. As a homing peptide, LyP-1 has shown properties of targeting, internalization, and proapoptosis to TNBC. In the study, we designed a Technetium-99m- ( 99m Tc-) labeled LyP-1 and investigated its feasibility for targeted single-positron emission computed tomography (SPECT) imaging of TNBC. The results showed that the LyP-1 peptide had acceptable biocompatibility in the studied concentration range and could specifically bind to TNBC cells in vitro. 99m Tc-labeled LyP-1 showed high radiochemical purity and stability and could be used as a probe for targeted SPECT imaging of TNBC cells in vitro and in a TNBC tumor-bearing mouse model. Our findings indicate that this active-targeted strategy has great potential to be developed into a new imaging tool for TNBC diagnosis., Competing Interests: The authors declare that there are no conflicts of interest., (Copyright © 2019 Ningning Song et al.)

Enumeration of tumor-infiltrating lymphocytes (TILs) in H&E stained tissue sections has demonstrated limited value in predicting immune responses to cancer immunotherapy, likely reflecting the diversity of cell types and immune activation states among tumor infiltrates. Multiparametric flow cytometry enables robust phenotypic and functional analysis to distinguish suppression from activation, but tissue dissociation eliminates spatial context. Multiplex methods for immunohistochemistry (IHC) are emerging, but these interrogate only a single tissue section at a time. Here, we report transparent tissue tomography (T3) as a tool for three-dimensional (3D) imaging cytometry in the complex architecture of the tumor microenvironment, demonstrating multiplexed immunofluorescent analysis in core needle biopsies. Using T3 imaging, image processing and machine learning to map CD3 + CD8 + cytotoxic T cells (CTLs) in whole core needle biopsies from Her2 + murine mammary tumors and human head and neck surgical specimens revealed marked inhomogeneity within single needle cores, confirmed by serial section IHC. Applying T3 imaging cytometry, we discovered a strong spatial correlation between CD3 + CD8 + CTLs and microvasculature in the EGFR + parenchyma, revealing significant differences among head and neck cancer patients. These results show that T3 offers simple and rapid access to three-dimensional and quantitative maps of the tumor microenvironment and immune infiltrate, offering a new diagnostic tool for personalized cancer immunotherapy.

Purpose: In order to monitor the drug responses of three-dimensional mammary tumor spheroids and to elucidate the role of inter- and intra-spheroid heterogeneity in determining drug sensitivity in the spheroids, an integrated image analysis framework was developed for morphometric and metabolic characterization of the three-dimensional tumor spheroids., Procedure: Three-dimensional spheroid cultures of primary mammary tumor epithelial cells isolated from freshly excised tumors from a transgenic mouse model of adenocarcinoma (MMTV-PyMT) were imaged by using vital dyes and mitochondrial membrane potential markers. Custom-developed java and python program codes facilitated image processing, numerical computation, and graphical analysis of large datasets generated from the experiments. A panel of cancer drugs (rapamycin, BEZ235, MK2206, and flavopiridol) was tested to determine the degree of drug sensitivity as well as heterogeneity in drug response., Results: A new quantitative metric (growth/toxicity) was developed based on morphometric parameters that were found to track the growth and apoptotic cell populations. Further, this study identified two parameters, namely, skew and kurtosis-which report the spatial heterogeneity in mitochondrial metabolism within the spheroids. The results of this study show that three-dimensional tumor spheroids selectively respond to cancer drugs depending on the specific metabolic pathways (AKT inhibition pathway in the present study), and there exists significant heterogeneity in the untreated tumor spheroids. Drug sensitivity of the spheroids was found to be associated with significant alterations in mitochondrial heterogeneity within the spheroids., Conclusions: In conclusion, the quantitative imaging of morphometric and metabolic analysis in large image datasets can serve as an excellent tool box for characterizing tumor heterogeneity in three-dimensional tumor spheroids and potentially, in intact tumors as well.

Purpose: Fluorescence lifetime imaging microscopy (FLIM) of endogenous fluorescent metabolites permits the measurement of cellular metabolism in cell, tissue and animal models. In parallel, magnetic resonance spectroscopy (MRS) of dynamic nuclear (hyper)polarized (DNP) 13 C-pyruvate enables measurement of metabolism at larger in vivo scales. Presented here are the design and initial application of a bioreactor that connects these 2 metabolic imaging modalities in vitro, using 3D cell cultures., Methods: The model fitting for FLIM data analysis and the theory behind a model for the diffusion of pyruvate into a collagen gel are detailed. The device is MRI-compatible, including an optical window, a temperature control system and an injection port for the introduction of contrast agents. Three-dimensional printing, computer numerical control machining and laser cutting were used to fabricate custom parts., Results: Performance of the bioreactor is demonstrated for 4 T1 murine breast cancer cells under glucose deprivation. Mean nicotinamide adenine dinucleotide (NADH) fluorescence lifetimes were 10% longer and hyperpolarized 13 C lactate:pyruvate (Lac:Pyr) ratios were 60% lower for glucose-deprived 4 T1 cells compared to 4 T1 cells in normal medium. Looking at the individual components of the NADH fluorescent lifetime, τ 1 (free NADH) showed no significant change, while τ 2 (bound NADH) showed a significant increase, suggesting that the increase in mean lifetime was due to a change in bound NADH., Conclusion: A novel bioreactor that is compatible with, and can exploit the benefits of, both FLIM and 13 C MRS in 3D cell cultures for studies of cell metabolism has been designed and applied., (© 2019 International Society for Magnetic Resonance in Medicine.)

Background: Tungsten disulfide (WS 2 ), which enjoyed a good potential to be a promising clinical theranostic agent for cancer treatment, is still subject to the tedious synthesis procedure., Methods: Here, we reported a one-pot 'bottom-up' hydrothermal strategy for the fabrication of PEGylated WS 2 nanoparticles (NPs). The WS 2 -PEG nanoparticles were characterized systematically. The CT imaging and photothermal therapy against tumor as well as biosafety in vitro and in vivo were also investigated., Results: The obtained WS 2 -PEG NPs enjoyed obvious merits of good solubility and favorable photothermal performance. WS 2 -PEG NPs exhibited desirable photothermal ablation ability against cancer cells and cancer cell-bearing mice in vitro and in vivo. MTT assay and histological analysis demonstrated the low cytotoxicity and biotoxicity of WS 2 -PEG NPs, providing a valid biosafety guarantee for the coming biomedical applications. In addition, thanks to the obvious X-ray attenuation of W atom, the WS 2 -PEG NPs can also be served as a favorable contrast agent for CT imaging of tumors., Conclusion: WS 2 -PEG NPs has enjoyed a good potential to be a promising clinical CT-guided photothermal therapeutic agent against cancers., (Copyright © 2019. Published by Elsevier Inc.)

The abnormal expression of tumor-related proteases plays a critical role in cancer invasion, progression, and metastasis. Therefore, it is considerably meaningful to non-invasively assess the proteases' activity in vivo for both tumor diagnosis and therapeutic evaluation. Herein, we report an activatable probe constructed with a near-infrared dye (Cy5.5) and a quencher (QSY21) covalently linked through a peptide substrate of matrix metalloproteinases-2 (MMP-2) that was chosen as a model for tumor-associated proteases. Upon cleavage with activated MMP-2, this probe emitted an MMP-2-concentration-dependent fluorescence. Quite unexpectedly, owing to the variation in the aggregation state of both the dye and its quencher as a consequence of the cleavage, the responsive probe presented a dramatic MMP-2-concentration-dependent absorption at around 680 nm, while that at around 730 nm was MMP-2 concentration independent. These features allowed detection of MMP-2 activity via both fluorescence and photoacoustic (PA) imaging in vitro, respectively. Moreover, taking the PA signal at 730 nm as an internal reference, the PA signal at 680 nm allowed quantitative detection of MMP-2 expression in breast cancer in vivo. We thus envision that our current approach would offer a useful tool for studying the malignant impacts of versatile tumor-associated proteases in vivo.

The overexpression of nitroreductase (NTR) in hypoxia has been recognized as a biomarker of highly aggressive disease, and the development of a hypoxia-sensitive two-photon (TP) bioimaging probe with both excitation and emission wavelengths in the red-light region provides favorable deep-tissue imaging with a low background fluorescence signal. Although quite a few TP hypoxia-sensitive fluorescent probes have been reported for NTR detection, their short emission wavelength (<550 nm) limits their application. Herein, we report a red light emissive TP hypoxia-sensitive turn-on probe (NRP) by employing Nile Red as a red-emitting fluorophore and p-nitrobenzene as an NTR recognition group with improved sensitivity. The NRP probe showed obvious strong red-fluorescence enhancement in the presence of NTR and high selectivity toward NTR in aqueous solution. Our in vitro experimental results illustrated that the NRP loaded tumor cells treated under hypoxia display remarkably strong fluorescence in both OP and TP microscopy at 655 nm with 45-fold enhancement, which affords deep-tissue penetration ability. The NRP probe was also successfully applied for imaging NTR in liver tissue slices and a 4T1-bearing mice model, which is important for bioimaging applications.

Macromolecular cancer drugs such as therapeutic antibodies and nanoparticles are well known to display slow extravasation and incomplete penetration into tumors, potentially protecting cancer cells from therapeutic effects. Conventional assays to track macromolecular drug delivery are poorly matched to the heterogeneous tumor microenvironment, but recent progress on optical tissue clearing and three-dimensional (3D) tumor imaging offers a path to quantitative assays with cellular resolution. Here, we apply transparent tissue tomography (T3) as a tool to track perfusion and delivery in the tumor and to evaluate target binding and vascular permeability. Using T3, we mapped anti-programmed cell death protein-ligand 1 (PD-L1) antibody distribution in whole mouse tumors. By measuring 3D penetration distances of the antibody drug out from the blood vessel boundaries into the tumor parenchyma, we determined spatial pharmacokinetics of anti-PD-L1 antibody drugs in mouse tumors. With multiplex imaging of tumor components, we determined the distinct distribution of anti-PD-L1 antibody drug in the tumor microenvironment with different PD-L1 expression patterns. T3 imaging revealed CD31 + capillaries are more permeable to anti-PD-L1 antibody transport compared with the blood vessels composed of endothelium supported by vascular fibroblasts and smooth muscle cells. T3 analysis also confirmed that isotype IgG antibody penetrates more deeply into tumor parenchyma than anti-Her2 or anti-EGFR antibody, which were restrained by binding to their respective antigens on tumor cells. Thus, T3 offers simple and rapid access to 3D, quantitative maps of macromolecular drug distribution in the tumor microenvironment, offering a new tool for development of macromolecular cancer therapeutics., (©2018 American Association for Cancer Research.)

Liposomes have been used as popular drug delivery systems for cancer therapy. However, it is difficult to track traditional liposome delivery systems in an efficient and stable fashion to assess their delivery efficacy and biodistribution after administration. Meanwhile, conventional fluorescent liposomes containing optical tracers face the challenge of aggregation-caused quenching. Herein, we report a strategy for the integration of an aggregation-induced emission fluorogen with a liposome to yield an AIEgen-lipid conjugate, termed "AIEsome". The AIEsome exhibits bright red fluorescence along with great photostability and biocompatibility, and can be used for in vitro cancer cell labeling and in vivo tumor targeting. Meanwhile, benefiting from the excellent photosensitizing ability of the AIEgen and its good oxygen exposure in aqueous media, the AIEsome also performs well in efficient photodynamic therapy (PDT) for both in vitro cancer cell ablation and in vivo antitumor therapy after white light illumination., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Free radicals have emerged as new-type and promising candidates for hypoxic tumor treatment, and further study of their therapeutic mechanism by real-time imaging is of great importance to explore their biomedical applications. Herein, we present a smart free-radical generator AuNC-V057-TPP for hypoxic tumor therapy; the AuNC-V057-TPP not only exhibits good therapeutic effect under both hypoxic and normoxic conditions but also can monitor the release of free radicals in real-time both in vitro and in vivo. What is more, with the mitochondria-targeting ability, the AuNC-V057-TPP is demonstrated with improved antitumor efficacy through enhanced free radical level in mitochondria, which leads to mitochondrial membrane damage and ATP production reduction and finally induces cancer cell apoptosis.

Photodynamic therapy (PDT) is a light-triggered therapy used to kill cancer cells by producing reactive oxygen species (ROS). Herein, a new kind of DNA nanostructure based on the coordination between calcium ions (Ca 2+ ) and AS1411 DNA G quadruplexes to form nanoscale coordination polymers (NCPs) is developed via a simple method. Both chlorine e6 (Ce6), a photosensitizer, and hemin, an iron-containing porphyrin, can be inserted into the G-quadruplex structure in the obtained NCPs. With further polyethylene glycol (PEG) modification, we obtain Ca-AS1411/Ce6/hemin@pHis-PEG (CACH-PEG) NCP nanostructure that enables the intranuclear transport of photosensitizer Ce6 to generate ROS inside cell nuclei that are the most vulnerable to ROS. Meanwhile, the inhibition of antiapoptotic protein B-cell lymphoma 2 (Bcl-2) expression by AS1411 allows for greatly improved PDT-induced cell apoptosis. Furthermore, the catalase-mimicking DNAzyme function of G-quadruplexes and hemin in those NCPs could decompose tumor endogenous H 2 O 2 to in situ generate oxygen so as to further enhance PDT by overcoming the hypoxia-associated resistance. This work develops a simple yet general method with which to fabricate DNA-based NCPs and presents an interesting concept of a nanoscale drug-delivery system that could achieve the intranuclear delivery of photosensitizers, the down-regulation of anti-apoptotic proteins, and the modulation of the unfavorable tumor microenvironment simultaneously for improved cancer therapy.

Hypoxia-induced radioresistance is the primary reason for failure of tumor radiotherapy (RT). Changes within the irradiated tumor microenvironment (TME) including oxygen, reactive oxygen species (ROS) and nitric oxide (NO) are closely related to radioresistance. Therefore, there is an urgent need to develop new approaches for overcoming hypoxic radioresistance by incorporating TME regulation into current radiotherapeutic strategies., Methods: Herein, we explored a radiation-responsive nanotheranostic system to enhance RT effects on hypoxic tumors by multi-way therapeutic effects. This system was developed by loading S-nitrosothiol groups (SNO, a NO donor) and indocyanine green (ICG, a photosensitizer) onto mesoporous silica shells of Eu 3+ -doped NaGdF 4 scintillating nanocrystals (NSC)., Results: Under X-ray radiation, this system can increase the local dosage by high-Z elements, promote ROS generation by X-ray-induced photodynamic therapy, and produce high levels of NO to enhance tumor-killing effects and improve hypoxia via NO-induced vasodilation. In vitro and in vivo studies revealed that this combined strategy can greatly reinforce DNA damage and apoptosis of hypoxic tumor cells, while significantly suppressing tumor growth, improving tumor hypoxia and promoting p53 up-regulation and HIF1α down-regulation. In addition, this system showed pronounced tumor contrast performance in T 1 -weighted magnetic resonance imaging and computed tomography., Conclusion: This work demonstrates the great potential of scintillating nanotheranostics for multimodal imaging-guided X-ray radiation-triggered tumor combined therapy to overcome radioresistance., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

IFNγ is an attractive target for imaging active antitumor immunity due to its function in the T-cell signaling axis. Here, we test an IFNγ immuno-PET (immunoPET) probe for its capacity to identify adaptive immunotherapy response after HER2/neu vaccination in both spontaneous salivary and orthotopic neu + mouse mammary tumors. IFNγ immunoPET detected elevated cytokine levels in situ after vaccination, which inversely correlated with tumor growth rate, an indicator of response to therapy. In a model of induced T-cell anergy where CD8 T cells infiltrate the tumor, but upregulate PD-1, IFNγ tracer uptake was equivalent to isotype control, illustrating a lack of antitumor T-cell activity. The IFNγ immunoPET tracer detected IFNγ protein sequestered on the surface of tumor cells, likely in complex with the IFNγ receptor, which may explain imaging localization of this soluble factor in vivo Collectively, we find that the activation status of cytotoxic T cells is annotated by IFNγ immunoPET, with reduced off-target binding to secondary lymphoid tissues compared with imaging total CD3 + tumor-infiltrating lymphocytes. Targeting of soluble cytokines such as IFNγ by PET imaging may provide valuable noninvasive insight into the function of immune cells in situ Significance: This study presents a novel approach to monitor therapeutic outcomes via IFNγ-targeted positron emission tomography. Cancer Res; 78(19); 5706-17. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Gold complexes can serve as efficient photothermal converters for cancer therapy, but their non-biodegradability hinders clinical bioapplications. Although enormous effort has been devoted, the conventionally adopted synthetic methods of biodegradation are characterized by high cost and complicated procedures, which delay the process of further clinical translation of gold complexes. Here, we report a multifunctional poly(amino acid)-gold-magnetic complex with self-degradation properties for synergistic chemo-photothermal therapy via simple and green chemistry methods. Nanoparticles of ∼3 nm in the biodegradation product were observed in simulated body fluid in 4 days. The biodegradability mainly benefits from the weakened internal electrostatic interaction of the poly(amino acid) by the ions in simulated body fluid. It is demonstrated that the poly(amino acid)-gold-magnetic complex has great cellular endocytosis by taking advantage of the guanidine group in arginine and possesses multimodal imaging and efficient tumor ablation (94%). This study reports a possibility for gold-magnetic complexes composed of poly(amino acid) to serve as a biodegradable nanotherapeutic for clinical applications.

Cancer invasion programs are adaptive by switching between metastatic collective and single-cell dissemination; however, current intravital microscopy models for epithelial cancer in mice fail to reliably recreate such invasion plasticity. Using microimplantation of breast cancer spheroids into the murine mammary fat pad and live-cell monitoring, we show microenvironmental conditions and cytoskeletal adaptation during collective to single-cell transition in vivo E-cadherin-expressing 4T1 and E-cadherin-negative MMT tumors both initiated collective invasion along stromal structures, reflecting invasion patterns in 3D organotypic culture and human primary ductal and lobular carcinoma. Collectively invading cells developed weakly oscillatory actin dynamics, yet provided zones for single-cell transitions with accentuated, more chaotic actin fluctuations. This identifies collective invasion in vivo as a dynamic niche and efficient source for single-cell release., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Background: The aim of this study was to evaluate the early anti-tumor efficiency of different therapeutic agents with a combination of multi-b-value DWI, DCE-MRI and texture analysis., Methods: Eighteen 4 T1 homograft tumor models were divided into control, paclitaxel monotherapy and paclitaxel and bevacizumab combination therapy groups (n = 6) that underwent multi-b-value DWI, DCE-MRI and texture analysis before and 15 days after treatment., Results: After treatment, the tumors in the control group were significantly larger than those in the combination group (P = 0.018). In multi-b-value DWI, the ADC slow obviously increased in the combination group compared to that in the others (P < 0.01). The f increased in the control and paclitaxel groups, but the combination group showed a significant decrease versus the others (P < 0.02). Additionally, in DCE-MRI, the decreasing K trans showed an evident difference between the combination and control groups (P = 0.003) due to the latter's increasing K trans . The intra-group comparisons of tumor texture in pre-, mid- and post-treatments showed that the entropy had all significantly increased in all groups (P < 0.01, SSF = 0-6), though the MPP, mean and SD increased only in the combination group (P MPP,mean,SD  < 0.05, SSF = 4-6). Moreover, the inter-group comparisons revealed that the mean and MPP exhibited significant differences after treatment (P mean,MPP  < 0.05, SSF = 0-3)., Conclusion: All these results suggest some strong correlations among DWI, DCE and texture analysis, which are beneficial for further study and clinical research.

Due to the importance of presurgical, noninvasive, and accurate diagnostic tools in mammary carcinoma characterization, this prospective secondary observational cohort study was designed to evaluate and compare the diagnostic accuracy of B-mode, Doppler, contrast enhancement ultrasonography, or acoustic radiation force impulse-elastography in identifying mammary carcinomas types with high degree of malignancy. A total of 246 mammary carcinomas from 141 female dogs were analyzed using B-mode, Doppler, contrast enhancement ultrasonography, and acoustic radiation force impulse ultrasonography prior to their histopathological classification according to types (simple, complex, or special) and grade (I, II, or III). Qualitative and quantitative variables were compared between carcinoma types and grades by Fisher's or analysis of variance. Diagnostic performance was estimated by receiver-operating characteristic analysis, using histopathological classification as a reference. Deformability (acoustic radiation force impulse) had a diagnostic specificity of 100% and sensitivity of 12% in identifying special carcinomas. A width:length ratio greater than 0.53 can be suggestive of special carcinoma, with 80% sensitivity and 76% specificity. Contrast wash-in and peak enhancement times lower than 7.5 and 13.5 s, respectively, were indicative of complex carcinoma at 62% sensitivity and 60% specificity. Contrast wash-in, peak enhancement, and wash-out times greater than 6.5, 12.5, and 64.5 s, respectively; were indicative of grade II and III carcinoma at 68% sensitivity and 62% specificity. In conclusion, B-mode ultrasonography, contrast enhancement ultrasonography, and acoustic radiation force impulse-elastography enabled the identification of some of the characteristics of high-grade mammary carcinoma types and grades in female dogs with limited accuracy. The findings from this study may contribute to oncology research and clinical management canine patients., (© 2018 American College of Veterinary Radiology.)

Ultrasonography is a valuable tool for the evaluation of neoplastic lesions in the dog and there is a growing interest in the use of this technique for the stadiation of canine mammary tumours. An accurate assessment of small sized nodules facilitates the stadiation of the mammary lesions and helps the clinician in the choice of the most indicated surgical therapy. The aim of this study was to identify those ultrasound criteria that may be useful in discriminating between benign and malignant lesions of small size (diameter smaller than 2 cm). Sixty-two nodules, < 2 cm in larger diameter, belonging to thirty-five bitches presented between January 2012 and February 2014 were evaluated. Tumours were observed by conventional ultrasound and assessed for: shape (regular-irregular), limit (defined-ill-defined), margins (regular-irregular), echogenicity (hypoechoic-isoechoic-hyperecoic), echotexture (homogeneus-heterogeneus), presence of hyperecoic halo, distal acoustic enhancement or shadowing and surrounding tissue alterations. Among the alterations in surrounding tissues, the disruption of the glandular tissue and the increase in echogenicity of the peritumoral tissues were assessed. Thereafter, bitches were subjected to mastectomy and nodules were evaluated histologically. None of the ultasound criteria considered in the current study showed a statistically significant relation with malignancy, except for the presence of alterations in the tissue surrounding the nodules. According to our results, this characteristic may indicate malignancy, however its subjectivity may affect the applicability in clinical practice. In conclusions, conventional ultrasound in bitches had a limited ability in discriminating benign and malignant mammary gland neoplastic lesions of small size (diameter < 2 cm).

Intravital microscopy (IVM) emerged and matured as a powerful tool for elucidating pathways in biological processes. Although label-free multiphoton IVM is attractive for its non-perturbative nature, its wide application has been hindered, mostly due to the limited contrast of each imaging modality and the challenge to integrate them. Here we introduce simultaneous label-free autofluorescence-multiharmonic (SLAM) microscopy, a single-excitation source nonlinear imaging platform that uses a custom-designed excitation window at 1110 nm and shaped ultrafast pulses at 10 MHz to enable fast (2-orders-of-magnitude improvement), simultaneous, and efficient acquisition of autofluorescence (FAD and NADH) and second/third harmonic generation from a wide array of cellular and extracellular components (e.g., tumor cells, immune cells, vesicles, and vessels) in living tissue using only 14 mW for extended time-lapse investigations. Our work demonstrates the versatility and efficiency of SLAM microscopy for tracking cellular events in vivo, and is a major enabling advance in label-free IVM.

The study objective was to investigate whether sex influences 3'-deoxy-3'- 18 F-fluorothymidine ( 18 F-FLT) uptake and tissue distribution in mouse models of cancer. Methods: 18 F-FLT biodistribution was measured in 3 strains of male and female mice (129S6/SvEv, athymic nude, and BALB/c). 18 F-FDG biodistribution was measured for comparison. 18 F-FLT uptake was also measured in female 129S6/SvEv mice bearing estrogen-dependent SSM3 mouse mammary tumors, male athymic nude mice bearing androgen-dependent CWR22 prostate cancer xenografts, and male and female athymic nude mice bearing estrogen-independent MDA-MB-231 human breast cancer xenografts. Ki-67 expression was assayed by immunohistochemistry. PET/CT imaging was performed to visualize 18 F-FLT biodistribution and to determine pharmacokinetics. Results: Greater 18 F-FLT activity was observed in blood, liver, muscle, heart, kidney, and bone in female than male mice. Pharmacokinetic analysis demonstrated higher early renal 18 F-FLT activity and greater accumulation of 18 F-FLT in the urinary bladder in male than female mice. The differential pattern of 18 F-FLT biodistribution between the sexes seen with 18 F-FLT was not observed with 18 F-FDG. Increased tumoral 18 F-FLT uptake compared with muscle was observed in both the SSM3 mammary tumors (2.4 ± 0.17 vs. 1.6 ± 0.14 percentage injected dose [%ID]/g at 2 h after injection, P = 0.006) and the CWR22 prostate cancer xenografts (0.34 ± 0.08 vs. 0.098 ± 0.033 %ID/g at 2 h after injection, P = 0.03). However, because of higher nonspecific muscle uptake in female mice, tumor-to-muscle uptake ratios were greater for CWR22 tumors than for SSM3 tumors (4.2 ± 0.78 vs. 1.5 ± 0.049 at 2 h after injection, P = 0.008). Sex-dependent differences in 18 F-FLT uptake were also observed for MDA-MB-231 xenografts (tumor-to-muscle ratio, 7.2 ± 0.9 for female vs. 16.9 ± 8.6 for male, P = 0.039). Conversely, greater tumoral Ki-67 staining was observed in female mice (71% ± 3% for female vs. 54% ± 2% for male, P = 0.009), and this finding more closely matched the relative differences in absolute 18 F-FLT tumor uptake values (4.5 ± 0.99 %ID/g for female vs. 1.9 ± 0.30 %ID/g for male, P = 0.03). Conclusion: Depending on whether female or male mice are used, differences in biodistribution and nonspecific tissue uptake can adversely affect quantitative measures of 18 F-FLT uptake. Thus, sex is a potential variable to consider in defining quantitative imaging metrics using 18 F-FLT to assess tumor proliferation., (© 2018 by the Society of Nuclear Medicine and Molecular Imaging.)

OBJECTIVE To evaluate the impact of 2 tidal volumes (T V s) with or without positive end-expiratory pressure (PEEP) on lung mechanics, aeration, and gas exchange in healthy anesthetized dogs. ANIMALS 40 mixed-breed dogs with healthy lungs. PROCEDURES Anesthetized dogs were randomly assigned to 4 groups (n = 10/group) with different ventilatory settings: T V of 8 mL/kg and PEEP of 0 cm H 2 O (low T V group), T V of 8 mL/kg and PEEP of 5 cm H 2 O (low T V plus PEEP group), T V of 15 mL/kg and PEEP of 0 cm H 2 O (high T V group), or T V of 15 mL/kg and PEEP of 5 cm H 2 O (high T V plus PEEP group). Expired CO 2 and respiratory rate were titrated on the basis of a predetermined stepwise protocol. Gas exchange, respiratory mechanics, and pulmonary aeration were evaluated by means of CT 30 minutes after starting mechanical ventilation at the assigned setting. RESULTS Partial pressures of arterial and expired CO 2 were higher in the low T V and low T V plus PEEP groups than in the high T V and high T V plus PEEP groups. Peak and plateau airway pressures were higher in the PEEP group than in the other groups. Static lung compliance was higher in the high T V plus PEEP group than in the low T V group. Relative percentages of atelectatic and poorly aerated lung were lower in the high T V plus PEEP group than in the other groups. Oxygenation was similar among groups. CONCLUSIONS AND CLINICAL RELEVANCE Differences in T V and PEEP application during mechanical ventilation may affect respiratory function in anesthetized dogs with healthy lungs. Ventilation with a T V of 15 mL/kg and PEEP of 5 cm H 2 O significantly improved lung compliance and reduced the amount of atelectatic and poorly aerated lung.

Nanoagents of integrating multiple imaging and therapeutic modalities have attracted tremendous attention for biomedical applications. Herein, we synthesize porous hollow Fe 3 O 4 as a theranostic agent for MRI and combined photothermal/chemo cancer therapy. The as-prepared porous iron oxide nanoagents allow for T 2 -weighted MR imaging. Interestingly, we demonstrate that the porous structure endows the nanoagents an outstanding photothermal property for cancer cell killing, in comparison with other types of iron oxide nanomaterials. Under the exposure of an NIR laser, the heat produced by porous Fe 3 O 4 can accelerate the release of the loaded drug (e.g., DOX) to enhance chemotherapeutic efficacy, promoting the ablation of cancer cells with synergistic photothermal/chemotherapy.

Aim: To evaluate the advantages of nanomaterial methoxy poly(ethylene glycol)-poly(lactide-co-glycolide) (mPEG-PLGA) encapsulated doxorubicin (D/DOX) and paclitaxel (T/TAX; mPEG-PLGA-DT) over free form of DOX and TAX (DOX/TAX)., Materials & Methods: Metabonomics was conducted to characterize the systemic metabolic response of allograft breast cancer model mice to mPEG-PLGA-DT and DOX/TAX treatments., Results: Breast tumor growth induced metabolic reprogram in serum and multiple organs. DOX/TAX treatment could ameliorate the elevated energy and nucleotides demands in some organs while mPEG-PLGA-DT treatment showed outstanding therapeutic outcomes in restoring the metabolic phenotypes of serum and kidney from tumor-bearing mice to the healthy state., Conclusion: This investigation proved the biological advantages of mPEG-PLGA-DT over DOX/TAX in molecular level through the comparison between their metabolic responses in vivo.

Metastasis is the main cause of death in people with cancer. Early diagnosis and targeted therapy for metastasis is crucial for the survival of the cancer patients. However, metastasis is hard to trace for its small size, dispersed distribution and unvascularized anatomy. Here we report a biomimetic persistent luminescent nanoplatform for noninvasive high-sensitive diagnosis and 808 nm laser controlled photodynamics assisted chemotherapy of metastasis. The nanoplatform is composed of a photosensitizer functionalized persistent luminescent nanoparticle core, a doxorubicin loaded hollow silica interlayer and a cancer cell membrane shell for effective metastasis theranostic. The cancer cell membrane shell prevents drug leakage and endows the nanoplatform with targeting ability to metastasis. The reactivatable persistent luminescence of persistent luminescent nanoparticles not only enables long-term in vivo metastasis tracking, but also provides internal light source for singlet oxygen generation to kill cancer cells and further break the membrane shell for drug release. This work provides a promising strategy to develop persistent luminescence imaging guided theranostic nanoplatforms for early metastasis.

Many cancers adeptly modulate metabolism to thrive in fluctuating oxygen conditions; however, current tools fail to image metabolic and vascular endpoints at spatial resolutions needed to visualize these adaptations in vivo. We demonstrate a high-resolution intravital microscopy technique to quantify glucose uptake, mitochondrial membrane potential (MMP), and SO 2 to characterize the in vivo phentoypes of three distinct murine breast cancer lines. Tetramethyl rhodamine, ethyl ester (TMRE) was thoroughly validated to report on MMP in normal and tumor-bearing mice. Imaging MMP or glucose uptake together with vascular endpoints revealed that metastatic 4T1 tumors maintained increased glucose uptake across all SO 2 ("Warburg effect"), and also showed increased MMP relative to normal tissue. Non-metastatic 67NR and 4T07 tumor lines both displayed increased MMP, but comparable glucose uptake, relative to normal tissue. The 4T1 peritumoral areas also showed a significant glycolytic shift relative to the tumor regions. During a hypoxic stress test, 4T1 tumors showed significant increases in MMP with corresponding significant drops in SO 2 , indicative of intensified mitochondrial metabolism. Conversely, 4T07 and 67NR tumors shifted toward glycolysis during hypoxia. Our findings underscore the importance of imaging metabolic endpoints within the context of a living microenvironment to gain insight into a tumor's adaptive behavior.

In recent years, phthalocyanines (Pcs) have been widely used as photosensitizer in photodynamic therapy applications. Because of their strong absorptions in the near-infrared region (640-700 nm). The integration of phthalocyanine derivatives to a nanoparticle is expected to be efficient way to improve the activity of the photosensitizer on the targeted tissue. It is known that the integrated molecules not only show better accumulation on tumor tissue but also reduce toxicity in healthy tissues. In this study, the ZnPc molecule was synthesized and integrated to the TiO 2 nanoparticle, to investigate the potential of PDT and its cytotoxicity. Additionally, ZnPc and ZnPc-TiO 2 molecules were labeled with 131 I and it was aimed to put forth the nuclear imaging/therapy potentials of 131 I labeled ZnPc/ZnPc-TiO 2 by determining in vitro uptakes in mouse mammary carcinoma (EMT6), human cervical adenocarcinoma (HeLa). In result of our study, it was observed that the radiolabeling yields of the synthesized ZnPc and ZnPc-TiO 2 with 131 I were quite high. In vitro uptake studies shown that 131 I-ZnPc-TiO 2 could be a potential agent for nuclear imaging/treatment of breast and cervical cancers. According to PDT results, ZnPc-TiO 2 might have as to be a potential PDT agent in the treatment of cervical tumor. ZnPc and ZnPc-TiO 2 might be used as theranostic agents., (© 2017 John Wiley & Sons A/S.)

Nanoparticles represent the most widely studied drug delivery systems targeting cancer. Polymeric nanoparticles can be easily generated through a microemulsion polymerization. Herein, the synthesis, radiolabeling, and in vivo evaluation of nanoparticles (NPs) functionalized by an organosilicon fluoride acceptor (SiFA) are reported which can be radiolabeled without further chemical modifications. Four nanoparticles in the sub-100 nm range with distinct hydrodynamic diameters of 20 nm (NP1), 33 nm (NP2), 45 nm (NP3), and 72 nm (NP4), respectively, were synthesized under size-controlled conditions. The SiFA-labeling building block acted as an initiator for the polymerization of polymer P1. The nanoparticles were radiolabeled with fluorine-18 ( 18 F) through simple isotopic exchange (IE) and analyzed in vivo in a murine mammary tumor model (EMT6). The facile 18 F radiolabeling SiFA methodology, performed in ethanol under mild reaction conditions, gave radiochemical yields (RCYs) of 19-26% and specific activities (SA) of 0.2-0.3 GBq/mg. Based on preclinical PET analysis, the tumor uptake and clearance profiles were analyzed depending on particle size. The nanoparticle size of 33 nm showed the highest tumor accumulation of SUV mean 0.97 (= 4.4%ID/g) after 4 h p.i. through passive diffusion based on the Enhanced Permeability and Retention (EPR) effect. Overall, this approach exhibits a simple, robust, and reliable synthesis of 18 F radiolabeled polymeric nanoparticles with a favorable in vivo evaluation profile. This approach represents a straightforward synthetically accessible alternative to produce radiolabeled nanoparticles without any further surface modification to attach a radioisotope.

Peptides are a growing class of macromolecules used in pharmaceutics. The path toward the clinical use of candidate peptides involves sequence optimization and cyclization for stability and affinity. For internalized peptides, tagging is also often required for intracellular trafficking studies, although fluorophore conjugation has an impact on peptide binding, permeability, and localization. Herein, a strategy based on cysteine arylation with tetrafluoroterephthalonitrile (4F-2CN), which simultaneously cyclizes peptides and imparts fluorescence, is reported. The 4F-2CN cyclization of an M2 macrophage-targeting peptide yields, in a single step, a peptide with improved serum stability, intrinsic fluorescence, and increased binding affinity. In a murine breast cancer model, it is demonstrated that the intrinsic fluorescence from the cyclized peptide is sufficient for monitoring biodistribution by whole-organ fluorescence imaging and cell internalization by flow cytometry., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Recent developments in optical tissue clearing and microscopic imaging have advanced three-dimensional (3D) visualization of intact tissues and organs at high resolution. However, to expand applications to oncology, critical limitations of current methods must be addressed. Here we describe transparent tissue tomography (T3) as a tool for rapid, three-dimensional, multiplexed immunofluorescent tumor imaging. Cutting tumors into sub-millimeter macrosections enables simple and rapid immunofluorescence staining, optical clearing, and confocal microscope imaging. Registering and fusing macrosection images yields high resolution 3D maps of multiple tumor microenvironment components and biomarkers throughout a tumor. The 3D maps can be quantitatively evaluated by automated image analysis. As an application of T3, 3D mapping and analysis revealed a heterogeneous distribution of programmed death-ligand 1 (PD-L1) in Her2 transgenic mouse mammary tumors, with high expression limited to tumor cells at the periphery and to CD31 + vascular endothelium in the core. Also, strong spatial correlation between CD45 + immune cell distribution and PD-L1 expression was revealed by T3 analysis of the whole tumors. Our results demonstrate that a tomographic approach offers simple and rapid access to high-resolution three-dimensional maps of the tumor immune microenvironment, offering a new tool to examine tumor heterogeneity.

Objectives: This study aimed to evaluate the impact of long-term exercise training on the vascularization of rat mammary tumors., Methods: Female rats were divided into 4 groups: N-methyl-N-nitrosourea (MNU) treated sedentary, MNU treated exercised, control sedentary, and control exercised. Tumor development was induced in the MNU groups by MNU administration. Exercised groups were trained for 35 weeks. Tumor vascularization was evaluated by pulsed Doppler and contrast-enhanced ultrasonography., Results: The pulsatility and resistive indices were slightly higher in the MNU sedentary group (P > .05). Mammary tumors mainly had centripetal and heterogeneous enhancement of the contrast, clear margins, and the presence of penetrating vessels. The MNU exercised group had a lower arrival time and time to peak and higher peak intensity, wash-in, and wash-out (P > .05). The area under the curve was similar between groups (P > .05)., Conclusions: The contrast-enhanced ultrasonographic study did not detect differences in mammary tumor vascularization between MNU sedentary and MNU exercised groups previously detected by power Doppler imaging, B-flow imaging, and immunohistochemistry., (© 2017 by the American Institute of Ultrasound in Medicine.)

Background: Targeting tumor vasculature with vascular disrupting agents (VDAs) results in substantial cell death that precede tumor shrinkage. Here, we investigate the potential of hyperpolarized magnetic resonance spectroscopy (HPMRS) to monitor early metabolic changes associated with VDA treatment., Methods: Mice bearing C3H mammary carcinomas were treated with the VDAs combretastatin-A4-phosphate (CA4P) or the analog OXi4503, and HPMRS was performed following [1- 13 C]pyruvate administration. Similarly, treated mice were positron emission tomography (PET) scanned following administration of the glucose analog FDG. Finally, metabolic imaging parameters were compared to tumor regrowth delay and measures of vascular damage, derived from dynamic contrast-agent enhanced magnetic resonance imaging (DCE-MRI) and histology., Results: VDA-treatment impaired tumor perfusion (histology and DCE-MRI), reduced FDG uptake, increased necrosis, and slowed tumor growth. HPMRS, revealed that the [1- 13 C]pyruvate-to-[1- 13 C]lactate conversion remained unaltered, whereas [1- 13 C]lactate-to-[ 13 C]bicarbonate (originating from respiratory CO 2 ) ratios increased significantly following treatment., Conclusions: DCE-MRI and FDG-PET revealed loss of vessel functionality, impaired glucose delivery and reduced metabolic activity prior to cell death. [1- 13 C]lactate-to-[ 13 C]bicarbonate ratios increased significantly during treatment, indicating a decline in respiratory activity driven by the onset of hypoxia. HPMRS is promising for early detection of metabolic stress inflicted by VDAs, which cannot easily be inferred based on blood flow measurements.

The purpose of this work was to study the anatomic and metabolic changes that occur with tumor progression, regression and recurrence in a switchable MYC-driven murine breast cancer model. Serial 1 H MRI and hyperpolarized [1- 13 C]pyruvate metabolic imaging were used to investigate the changes in tumor volume and glycolytic metabolism over time during the multistage tumorigenesis. We show that acute de-induction of MYC expression in established tumors results in rapid tumor regression and significantly reduced glycolytic metabolism as measured by pyruvate-to-lactate conversion. Moreover, cancer recurrences occurring at the tumor sites independently of MYC expression were observed to accompany markedly increased lactate production., (Copyright © 2017 Elsevier Inc. All rights reserved.)

High animal fat consumption is associated with an increase in triple-negative breast cancer (TNBC) risk. Based on previous MRI studies demonstrating the feasibility of detecting very early non-palpable mammary cancers in simian virus 40 large T antigen (SV40TAg) mice, we examined the effect of dietary fat fed from weaning to young adulthood in this model of TNBC. Virgin female C3(1)SV40TAg mice (n = 16) were weaned at 3-4 weeks of age and then fed either a low fat diet (LFD) (n = 8, 3.7 kcal/g; 17.2% kcal from vegetable oil) or a high animal fat diet (HAFD) (n = 8, 5.3 kcal/g; 60% kcal from lard). After 8 weeks on the diet (12 weeks of age), fast spin echo MR images of inguinal mammary glands were acquired at 9.4 T. Following in vivo MRI, mice were sacrificed and inguinal mammary glands were excised and formalin fixed for ex vivo MRI. 3D volume-rendered MR images were then correlated with mammary gland histology to assess the glandular parenchyma and tumor burden. Using in vivo MRI, an average of 3.88 ± 1.03 tumors were detected per HAFD-fed mouse compared with an average of 1.25 ± 1.16 per LFD-fed mouse (p < 0.007). Additionally, the average tumor volume was significantly higher following HAFD feeding (0.53 ± 0.45 mm 3 ) compared with LFD feeding (0.20 ± 0.08 mm 3 , p < 0.02). Analysis of ex vivo MR and histology images demonstrated that HAFD mouse mammary glands had denser parenchyma, irregular and enlarged ducts, dilated blood vessels, increased white adipose tissue, and increased tumor invasion. MRI and histological studies of the SV40TAg mice demonstrated that HAFD feeding also resulted in higher cancer incidence and larger mammary tumors. Unlike other imaging methods for assessing environmental effects on mammary cancer growth, MRI allows routine serial measurements and reliable detection of small cancers as well as accurate tumor volume measurements and assessment of the three-dimensional distribution of tumors over time., (Copyright © 2017 John Wiley & Sons, Ltd.)

Increasing evidence indicates that the tumor microenvironment plays a critical role in regulating the biologic behavior of breast cancer. In veterinary oncology, there is a need for improved prognostic markers to accurately identify dogs at risk for local and distant (metastatic) recurrence of mammary gland carcinoma and therefore would benefit from adjuvant therapy. Collagen density and fiber organization have been shown to regulate tumor progression in both mouse and human mammary tumors, with certain collagen signatures predicting poor outcomes in women with breast cancer. We hypothesized that collagen signatures in canine mammary tumor biopsies can serve as prognostic biomarkers and potential targets for treatment. We used second harmonic generation imaging to evaluate fibrillar collagen density, the presence of a tumor-stromal boundary, tumor associated collagen signatures (TACS) and individual collagen fiber characteristics (width, length and straightness) in grade I/II and grade III canine mammary tumors. Collagen density, as well as fiber width, length and straightness, were inversely correlated with patient overall survival time. Notably, grade III cases were less likely to have a tumor-stromal boundary and the lack of a boundary predicted poor outcome. Importantly, a lack of a defined tumor-stromal boundary and an increased collagen fiber width were associated with decreased survival even when tumor grade, patient stage, ovariohysterectomy status at the time of mammary tumor excision, and histologic evidence of lymphovascular invasion were considered in a multivariable model, indicating that these parameters could augment current methods to identify patients at high risk for local or metastatic progression/recurrence. Furthermore, these data, which identify for the first time, prognostic collagen biomarkers in naturally occurring mammary gland neoplasia in the dog, support the use of the dog as a translational model for tumor-stromal interactions in breast cancer.