Triple-negative breast cancer (TNBC), as one of the most aggressive forms of breast cancer, is characterized by a poor prognosis and a very low rate of disease-free and overall survival. In recent years, immunotherapeutic approaches targeting T cell checkpoint molecules, such as cytotoxic lymphocyte antigen-4 (CTLA-4), programmed death1 (PD-1) or its ligand, programmed death ligand 1 (PD-L1), have shown great potential and have been used to treat various cancers as single therapies or in combination with other modalities. However, despite this remarkable progress, patients with TNBC have shown a low response rate to this approach, commonly developing resistance to immune checkpoint blockade, leading to treatment failure. Extracellular acidosis within the tumor microenvironment (also known as the Warburg effect) is one of the factors preventing immune cells from mounting effective responses and contributing to immunotherapy treatment failure. Therefore, reducing tumor acidity is important for increasing cancer immunotherapy effectiveness and this has yet to be realized in the TNBC environment. In this study, the oral administration of sodium bicarbonate (NaHCO3) enhanced the antitumor effect of anti-PD-L1 antibody treatment, as demonstrated by generated antitumor immunity, tumor growth inhibition and enhanced survival in 4T1-Luc breast cancer model. Here, we show that NaHCO3 increased extracellular pH (pHe) in tumor tissues in vivo, an effect that was accompanied by an increase in T cell infiltration, T cell activation and IFN-γ, IL2 and IL12p40 mRNA expression in tumor tissues, as well as an increase in T cell activation in tumor-draining lymph nodes. Interestingly, these changes were further enhanced in response to combined NaHCO3 + anti-PD-L1 therapy. In addition, the acidic extracellular conditions caused a significant increase in PD-L1 expression in vitro. Taken together, these results indicate that alkalizing therapy holds potential as a new tumor microenvironment immunomodulator and we hypothesize that NaHCO3 can enhance the antitumor effects of anti-PD-L1 breast cancer therapy. The combination of these treatments may have an exceptional impact on future TNBC immunotherapeutic approaches by providing a powerful personalized medicine paradigm. Therefore, our findings have a great translational potential for improving outcomes in TNBC patients.

Purpose/Objective(s)Triple negative breast cancer (TNBC) is the most aggressive breast cancer (BC) form, with a high metastases rate and a very low survival. The aggressiveness of TNBC coupled with a significant toxicity and suboptimal chemotherapy outcomes underscores the urgency for new TNBC treatments. In recent years, immunotherapy has emerged as a promising option. In particular, immune checkpoint blockers (ICB) targeting PD-L1/PD1 inhibitory T cell check point pathway showed clinical responses and have been explored for TNBC. Unfortunately, the response rates to standalone ICB therapy are low (15-20%), indicating the presence of inhibitory immune mechanisms. Radiation therapy (RT) has been widely used in BC therapies. In addition to antitumor (antiproliferative) effects, RT has been evidenced to stimulate immune tumor rejection through immunomodulation of the tumor microenvironment (TME) that has been shown to enhance the response to immunotherapy in mouse BC models. Antitumor RT effects, including TME immunomodulation, can be improved by using radiosensitizers, such as gold nanoparticles (AuNPs). We hypothesize that AuNP potentiates RT-induced immunomodulatory effects, leading to a more efficient response to ICB in TNBC. To test this hypothesis, we used AuNP as an enhancer of RT-induced immunological TME changes, to improve ICB therapy response in murine orthotopic syngeneic 4T1Luc TNBC model.Materials/MethodsFemale Balb/c mice bearing 4T1Luc tumors received intratumoral injections of 14 nm AuNPs. After 24h mice were irradiated with fractionated regimen of 3 × 6 Gy dose using 225 kV photons. After the 3rd RT dose, mice received 3 doses of anti-PD-L1 antibody that were 4 days apart. Therapeutic efficiency was determined by assessing the tumor growth and animal survival. Tumor tissue immunohistochemistry determined the expression of TME immunological markers and immune cell tumor infiltration.ResultsAuNPs improved response to anti PD-L1 treatment in mice receiving RT, shown by significant delay in tumor growth and increase in survival compared to the animals receiving RT+ AuNP (p<0.01) and to the animals receiving RT+ anti PD-L1 or RT alone (p<0.05). These results were accompanied with changes in the expression of TME immunological markers and T cell and macrophage infiltration.ConclusionIn TNBC patients, induction of antitumor immune response may play a critical role in improving clinical outcomes. Here we show that AuNP enhanced the effect of a fractionated RT regimen that has significantly improved the response to anti PD-L1 treatment in 4T1Luc TNBC mouse model. This effect was measured by a delay in tumor growth and an increase in animal survival. These findings support the role of immunological mechanisms in TNBC and provide a platform for designing multimodal TNBC RT formulations with novel radiosensitizers or immunotherapy.

Gold nanoparticles (AuNPs) have been shown to enhance cancer radiotherapy (RT) gain by localizing the absorption of radiation energy in the tumor while sparing surrounding normal tissue from radiation toxicity. Previously, we showed that AuNPs enhanced RT induced DNA damage and cytotoxicity in MCF7 breast cancer cells. Interestingly, we found that cancer cells exhibited a size-dependent AuNPs intracellular localization (4 nm preferentially in the cytoplasm and 14 nm in the nucleus). We extended those studies to an in vivo model and examined the AuNPs effects on RT cytotoxicity, survival and immunomodulation of tumor microenvironment (TME) in human triple negative breast cancer (TNBC) xenograft mouse model. We also explored the significance of nanoparticle size in these AuNPs' effects. Mice treated with RT and RT plus 4 nm or 14 nm AuNPs showed a significant tumor growth delay, compared to untreated animals, while dual RT plus AuNPs treatment exhibited additive effect compared to either RT or AuNPs treatment alone. Survival log-rank test showed significant RT enhancement with 14 nm AuNP alone; however, 4 nm AuNPs did not exhibit RT enhancement. Both sizes of AuNPs enhanced RT induced immunogenic cell death (ICD) that was coupled with significant macrophage infiltration in mice pretreated with 14 nm AuNPs. These results showing significant AuNP size-dependent RT enhancement, as evident by both tumor growth delay and overall survival, reveal additional underlying immunological mechanisms and provide a platform for studying RT multimodal approaches for TNBC that may be combined with immunotherapies, enhancing their effect.

Tumor immunogenic cell death (ICD) plays a major role in stimulating host anti-cancer immune response that can determine the success of cancer radiotherapy (RT). Additionally, ICD can also be induced by radiation itself. The main feature of ICD is the release of immunogenic molecules by dying cells, termed damage associated molecular patterns (DAMPs) that act on innate and adaptive immune components to induce long-lasting antitumor immunity. Two breast cancer (BCa) DAMPs involved in phagocytosis and dendritic cell antigen presentation are Calreticulin (CLR) and HMGB1; loss of HMGB1 expression is linked to poor clinical prognosis. Therefore, these molecules are being studied for their therapeutic and prognostic potential. As RT, either alone or in combination, is often part of standard BCa therapies, the effect of RT and radiosensitizers, such as gold nanoparticles (AuNPs), on DAMPs expression must be considered when designing new protocols, especially if combining RT with an adjuvant mode such as immunotherapy. The goal of this study was to measure the effect of radiation on CLR and HMGB-1 expression in the presence and absence of a novel AuNP radiosensitizer in MDA MB 231 BCa in vitro and in vivo models. We hypothesized that AuNP modulates radiation induced ICD by altering CLR and HMGB1 expression. In vitro: MDA MB 231 cells were incubated for 3h with 14nm AuNPs and irradiated. After 48h and 72h the percentages of CLR and HMGB1 positive cells were determined by flow cytometry. In vivo: Female nude mice bearing MDA MB 231 tumors received intratumoral injections of 14 nm AuNPs. After 24h mice were irradiated with 15 Gy dose using 160 kV photons. Mice were euthanized, histological sections prepared, stained with anti CLR antibodies and analyzed by light microscopy. In vitro, radiation induced increase in the percentages of CLR and HMGB1 positive cells was potentiated by AuNP at 48h and 72h time points. In control cells, AuNP alone increased the percent of HMGB1 positive cells at both time points. The percent of CLR positive cells was increased at 72h only. In vivo, in animals receiving RT or AuNP only, CLR expression was increased relative to control animals and CLR was detected extracellularly after RT, and extra- and intracellularly after AuNP alone. However, after combined RT and AuNP, CLR expression was higher than after single treatments and was detected both, extra- and intracellularly. In BCa patients, induction of ICD may play a critical role in improving clinical outcomes. Here we show that AuNP enhanced the immunogenic effect of a single irradiation dose in BCa cells and mouse model. This effect was measured by an increase in the expression of CLR and HMGB1, DAMPs critical to the response of BCa. In addition, AuNP alone exhibited an immunogenic effect similar to RT alone. These findings support that BCa depends on ICD and provide a platform for designing multimodal BCa RT formulations with novel radiosensitizers or immunotherapy.

PURPOSE:Deep learning models have had a great success in disease classifications using large data pools of skin cancer images or lung X-rays. However, data scarcity has been the roadblock of applying deep learning models directly on prostate multiparametric MRI (mpMRI). Although model interpretation has been heavily studied for natural images for the past few years, there has been a lack of interpretation of deep learning models trained on medical images. In this paper, an efficient convolutional neural network (CNN) was developed and the model interpretation at various convolutional layers was systematically analyzed to improve the understanding of how CNN interprets multimodality medical images and the predictive powers of features at each layer. The problem of small sample size was addressed by feeding the intermediate features into a traditional classification algorithm known as weighted extreme learning machine (wELM), with imbalanced distribution among output categories taken into consideration.METHODS:The training data collection used a retrospective set of prostate MR studies, from SPIE-AAPM-NCI PROSTATEx Challenges held in 2017. Three hundred twenty biopsy samples of lesions from 201 prostate cancer patients were diagnosed and identified as clinically significant (malignant) or not significant (benign). All studies included T2-weighted (T2W), proton density-weighted (PD-W), dynamic contrast enhanced (DCE) and diffusion-weighted (DW) imaging. After registration and lesion-based normalization, a CNN with four convolutional layers were developed and trained on tenfold cross validation. The features from intermediate layers were then extracted as input to wELM to test the discriminative power of each individual layer. The best performing model from the tenfolds was chosen to be tested on the holdout cohort from two sources. Feature maps after each convolutional layer were then visualized to monitor the trend, as the layer propagated. Scatter plotting was used to visualize the transformation of data distribution. Finally, a class activation map was generated to highlight the region of interest based on the model perspective.RESULTS:Experimental trials indicated that the best input for CNN was a modality combination of T2W, apparent diffusion coefficient (ADC) and DWIb50 . The convolutional features from CNN paired with a weighted extreme learning classifier showed substantial performance compared to a CNN end-to-end training model. The feature map visualization reveals similar findings on natural images where lower layers tend to learn lower level features such as edges, intensity changes, etc, while higher layers learn more abstract and task-related concept such as the lesion region. The generated saliency map revealed that the model was able to focus on the region of interest where the lesion resided and filter out background information, including prostate boundary, rectum, etc. CONCLUSIONS: This work designs a customized workflow for the small and imbalanced dataset of prostate mpMRI where features were extracted from a deep learning model and then analyzed by a traditional machine learning classifier. In addition, this work contributes to revealing how deep learning models interpret mpMRI for prostate cancer patient stratification.

In recent years, radiation therapy (RT) has been evidenced to stimulate immune tumor rejection through immunomodulation of the tumor microenvironment (TME). One of the RT immunomodulation mechanisms include immunogenic cell death (ICD) that plays a major role in stimulating host anti-cancer immune response and can determine the success of cancer RT. The main feature of ICD is the release of immunogenic molecules by dying cells, termed damage associated molecular patterns (DAMPs) that act on innate and adaptive immune components to induce long-lasting antitumor immunity. Calreticulin (CRT) is a DAMPs molecule involved in phagocytosis and dendritic cell antigen presentation. In breast cancer (BC) calreticulin pronounced expression was associated with tumor metastatic potential and size. Therefore, DAMPs are being studied for their therapeutic and prognostic potential. As RT, either alone or in combination, is often part of standard BC therapies, the effect of RT and radiosensitizers (such as gold nanoparticles (AuNP)) on DAMPs expression must be considered when designing new protocols, especially if combining RT with an adjuvant mode such as immunotherapy. The goal of this study was to measure the effect of radiation on CRT expression and associated macrophage infiltration in the presence and absence of a novel AuNP radiosensitizer in MDA MB 231 BC mouse models. We hypothesize that AuNP modulates RT induced immunological changes such as increase in CRT expression and infiltration by F4/80 positive macrophages. Female nude mice bearing MDA MB 231 tumors received intratumoral injections of 4nm or 14 nm AuNPs. After 24h mice were irradiated with 15 Gy dose using 160 kV photons. Mice were euthanized, histological sections prepared, stained with anti CRT and anti F4/80 antibodies and analyzed by light microscopy. In animals receiving RT or 14 nm AuNP only, CRT and F4/80 expression exhibited trend in increase relative to control but did not reach the significance. However, after the combined RT and AuNP (4nm or 14nm) treatment, CRT expression was further increased and reached the significance, compared to controls. However, F4/80 expression was significantly increased only in animals receiving the combination of RT and 14 nm AuNP, compared to controls (Table 1). In BC patients, induction of ICD may play a critical role in improving clinical outcomes. Here we show that AuNP enhanced the immunogenic effect of a single RT dose in BC mouse model. This effect was measured by an increase in the expression of CRT and F4/80, an indicator of macrophage infiltration. These findings support the role of immunological mechanisms in BC depends and provide a platform for designing multimodal BC RT formulations with novel radiosensitizers or immunotherapy.Abstract 3252; Table% positive staincontrolRT4 nm AuNP14 nm AuNPRT + 4 nm AuNPRT + 14 nm AuNPCalreticulin3.549.714.5612.6219.22*17.42*F4/8019.4422.4917.4520.4721.2030.99**p<0.05 compared to control. Open table in a new tab

Gold nanoparticles (AuNPs) are explored as radiosensitization probes enhancing cancer radiation treatment effects. Our long range goal is to fully characterize and optimize parameters for AuNPs use in cancer radiotherapy. We aim to develop AuNP probes that can be delivered to the cancer cells with the purpose of increasing their radiation sensitivity, and that would facilitate targeted delivery of high radiation doses to cancer, while sparing surrounding normal tissue from radiation toxic side effects.

Purpose: The aim of this study was to identify and rank discriminant radiomics features extracted from MR multi-modal images to construct an adaptive model for characterization of Dominant Intra-prostatic Lesions (DILs) from normal prostatic gland tissues (NT). Methods and Materials: Two cohorts were retrospectively studied: Group A consisted of 98 patients and Group B 19 patients. Two image modalities were acquired using a 3.0T MR scanner: Axial T2 Weighted (T2W) and axial diffusion weighted (DW) imaging. A linear regression method was used to construct apparent diffusion coefficient (ADC) maps from DW images. DILs and the NT in the mirrored location were drawn on each modality. One hundred and sixty-eight radiomics features were extracted from DILs and NT. A Partial-Least-Squares-Correlation (PLSC) with one-way ANOVA along with bootstrapping ratio techniques were recruited to identify and rank the most discriminant latent variables. An artificial neural network (ANN) was constructed based on the optimal latent variable feature to classify the DILs and NTs. Nineteen patients were randomly chosen to test the contour variability effect on the radiomics analysis and the performance of the ANN. Finally, the trained ANN and a two dimension (2D) convolutional sampling method were combined and used to estimate DIL-NT probability map for two test cases. Results: Among 168 radiomics-based latent variables, only the first four variables of each modality in the PLSC space were found to be significantly different between the DILs and NTs. Area Under Receiver Operating Characteristic (AUROC), Positive Predictive and Negative Predictive values (PPV and NPV) for the conventional method were 94%, 0.95, and 0.92, respectively. When the feature vector was randomly permuted 10,000 times, a very strong permutation-invariant efficiency (p < 0.0001) was achieved. The radiomic-based latent variables of the NTs and DILs showed no statistically significant differences (Fstatistic < Fc = 4.11 with Confidence Level of 95% for all 8 variables) against contour variability. Dice coefficients between DIL-NT probability map and physician contours for the two test cases were 0.82 and 0.71, respectively. Conclusion: This study demonstrates the high performance of combining radiomics information extracted from multimodal MR information such as T2WI and ADC maps, and adaptive models to detect DILs in patients with PCa.

Cell surface molecules expressed on cancer cells can be used as diagnostic and therapeutic tools. In breast cancer, CD44 and CD24 were identified as cell surface markers characterizing cancer cell stemness that may correlate with prognosis. Breast cancer cells with high CD44 and low CD24 expression have been shown to exhibit proliferative, invasive and metastatic properties that may relate to drug sensitivity and metastatic risk in patients. In addition to stemness, immune evasion is another hallmark of cancer with CD47 “don’t eat me” and CD274 “don’t find me” molecules playing important roles in antitumor immunity. Although each of these molecules are of interest as therapeutic targets, current therapeutic paradigms involve multimodal approach with radiation therapy often having a central role. Hence, the effect of irradiation, and related radio-sensitizers, on the expression of cancer cells surface markers to be targeted with an adjuvant therapeutic mode must be considered when designing such therapies. The goal of this study was to determine the dynamics of CD44, CD24, CD47 and CD274 expression in radio-sensitized and irradiated MDA-MB 231 breast cancer cells. We have recently reported that gold nanoparticles (AuNPs) sensitized breast cancer cells to irradiation at kV and MV energies. Here, we hypothesize that AuNP may also modulate the irradiation altered expression of cell surface markers. We explored this by using 10 MV energies and 6 Gy radiation dose. MDA-MB-231 cells were incubated for 3 hours with 14nm AuNPs, irradiated and allowed to grow for 24h and 72h after which percenteges of positive cells were determined by flow cytometry. Results are expressed as a percent of control, non-irradiated cells that was set at 100%. At 24h non-irradiated cells pre-incubated with AuNP exhibited a decrease in the percentage of CD24+ and CD44+ cells, while no significant change in the percentage of CD47+ and CD274+ cells was observed. Irradiation with 6Gy at 10 MV induced a decrease in CD24+ and an increase in CD44+ cells and AuNP potentiated this effect. However, 72h post-irradiation, percentages of CD24+ and CD44+ cells significantly increased, compared to non-irradiated controls and this effect was of lesser magnitude in AuNP pretreated cells. Percentage of CD47+ and CD274+ cells also significantly increased 24h post-irradiation, but with less magnitude in AuNP treated cells. At 72h in AuNP treated irradiated cells no change was observed in the percentage of CD274+ cells, while the percentage of CD47+ cells significantly increased in comparison to the irradiated cells not exposed to AuNPs. Preliminary data shown here indicate that expression of molecules important for cancer progression, metastasis and immune evasion undergo changes in response to irradiation, and that these changes are affected by AuNPs. Further studies will shed more light on the mechanisms behind these observed effects. Citation Format: Branislava Janic, Fangchao Liu, Kevin Bobbitt, Stephen Brown, Guangzhao Mao, Indrin Chetty, Benjamin Movsas, Ning Wen. Effect of irradiation and gold nanoparticle on expression dynamics of cell surface markers in MDA-MB 231 breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4862.

Gold nanoparticles (AuNPs) are shown to increase cancer cells sensitization to ionizing radiation.Theoretical models predicted that due to the differences in absorption coefficient between gold and soft tissue, maximum sensitization can be achieved at kilovoltage (kV), with minimum to no effect at megavoltage (MV) range, which was indeed demonstrated by previous research on AuNPs radio-sensitization with kV radiation.However, AuNPs effect at MV energies is still not clear.Radio-sensitization depends on AuNPs' internalization by cancer cells that is in turn affected by AuNPs size.The purpose of this study was to explore, in vitro, radio sensitization potential of AuNPs of 4 and 14 nm size with clinically relevant MV, as well as kV photons in MCF7 breast cancer cell line.We investigated the differences in cellular uptake and intracellular localization between 4 and 14 nm AuNPs by transmission electron microscopy (TEM).We also examined the effect of these AuNPs on MCF7 cells response to irradiation by MTT analysis of cell proliferation, and flow cytometry analysis of H2AX phosphorylation as an indicator of DNA double strand breaks (DSBs).Our results demonstrated significant AuNPs cellular uptake and a selective, size dependent intracellular localization with 4 nm AuNPs mostly residing in cytoplasm and 14 nm in nucleus.Nanoparticles of both sizes sensitized MCF-7 breast cancer cells to radiation at both kV and MV energies, independent of particle size.Radio-sensitization was detected as an increase in radiation induced inhibition of cell proliferation and radiation induced DNA DSBs.This work provides valuable information for further exploring mechanisms of AuNPs radio-sensitization in cancer cell lines that may be important in designing and optimizing AuNPs based radiotherapies.

Nanoparticles (NPs) are 1-100 nm constructs explored for their application in cancer diagnosis and treatment. Exposure of cells to ionizing irradiation leads to DNA damage, with DNA double strand breaks (DSBs) being the most toxic that can lead to apoptosis. The presence of gold in NPs enhances radiation damage and DSBs, the latter measured by the levels of phosphorylated DNA histone protein H2AX (γH2AX). The goal of this study was to decipher biological mechanisms of NP radio-sensitization. We analyzed the effects of two different sizes of gold nanoparticles (AuNP) on DSBs in MCF-7 breast cancer cells by assessing H2AX phosphorylation at three photon energies. Cells were incubated with either 4 nm or 14 nm AuNP and irradiated with 2, 4 or 8 Gy using 2.5 FFF MV (60 MU/min), 6 MV (600 MU/min) or 10 FFF MV (2400 MU/min) X-rays. Percent of cells positive for γH2AX was determined by flow cytometry. Live cells (100000) were gated using forward (FSC; cell size) versus side scatter (SSC; complexity) characteristics (gate P1). Cells positive for γH2AX were further gated to identify subpopulations exhibiting brighter (gate P2) or dimmer (gate P3) fluorescence intensity. Treatment with 4 nm AuNP resulted in significantly more γH2AX positive cells after irradiation at all three energies, compared to their respective controls. When treated at 6 MV energy, increases in the percentage of cells positive for H2AX phosphorylation was detected at all three doses (2, 4 and 8 Gy). Increased phosphorylation was accompanied by an increase of cells in the P2 gate accompanied by an increase in their FCS and SSC characteristics, consistent with activation. In cells treated with 10 MV the effect was most pronounced at 4Gy dose, while 2 and 8 Gy resulted in a slight increase in the percentage of γH2AX positive cells. Nevertheless, these cells still exhibited prominent increases within the P2 gate and FCS vs SSC characteristics, compared to their irradiated controls. In cells treated with 2.5 MV energy similar results were observed using 4 and 8 Gy, while no effect was detected with 2 Gy. Data from the experiments using 14 nm size AuNP were less clear and require further investigation. The difference may be due to the localization of the smaller NPs within the nucleus and therefore cause more DNA damage thereby greater H2AX phosphorylation. We hypothesize that the NP radio-sensitization mechanism involves directing cells towards apoptosis by enhancing DNA damage and interfering with DNA repair. Hence, γH2AX positive cells exhibiting bright fluorescence (P2) and an increase in FCS and SSC identified even in the conditions without significant increase in the percent of total γH2AX positive cells, may reflect cells primed for apoptosis. Future studies are planned to elucidate the exact intracellular mechanisms of NP biological radio-sensitization effect. Citation Format: Branislava Janic, Fangchao Liu, Kevin Bobbitt, Stephen Brown, Guangzhao Mao, Indrin J. Chetty, Benjamin Movsas, Ning Winston Wen. Effect of gold nanoparticle on radiation induced DNA damage in MCF7 breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1376.

Abstract Purpose: We investigated radiomic features extracted from dominant intraprostatic lesions (DILs) of the peripheral zone (PZ) and central gland (CG) from MR multi-modal images of 20 patients with prostate cancer (PCa). Remaining prostate gland (RPG) were included in the analysis. Material and Methods: 20 biopsy-proven PCa patients with no prior radiation treatment were studied. Axial T2 weighted images (T2WI) and diffusion weighted images (DWI) were acquired of the pelvis using a 3T MR scanner. ADC maps were constructed from DWIs. Region of interests delineating DILs and RPGs were contoured on each MR modality. 168 radiomic features were extracted from DIL and RPG volumes (15 pairs from PZ and 5 pairs from CG). Radiomic features were categorized into 8 different sets: Intensity Based Histogram (IBH, 9 features), Gray Level Run Length (GLRL, 7 features), Law's Textural Information (LAWS, 18 features), Discrete Orthonormal Stockwell Transform (DOST, 18 features), Local Binary Pattern (LBP, 6 features), 2D Wavelet Transform (2DWT, 48 features), 2D Gabor Filter (2DGF, 40 features), and Gray Level Co-Occurrence Matrix (GLCM, 22 features). ANOVA (with Bonferroni adjustment), overall mean percent difference (OMPD), and the Fisher combined probability were used to test the following 7 hypotheses: (1) DILs of PZ and CG from T2WI (2) DILs of PZ and CG from ADC (3) DILs and RPGs of PZ from T2WI (4) DILs and RPGs of PZ from ADC (5) DILs and RPGs of CG from T2WI (6) DILs and RPGs of CG from ADC (7) DILs of PZ for T2WI and ADC. Results: Results imply that among 168 radiomics features, only 5 (DOST, and 2DGF, OMPD=107.8%), 2 (2DWT, and 2DGF, OMPD=141.7%), 13 (IBH, 2DWT, and GLCM, OMPD=%226.6), 17 (IBH, 2DWT, and GLCM, OMPD=179.7%), 18 (IBH, 2DWT, and GLCM, OMPD=321.9%), 18 (IBH, 2DWT, and GLCM, OMPD=726.1%), and 74 (IBH, GLRL, LAWS, DOST, LBP, 2DWT, 2DGF, GLCM, OMPD=1564%) features are discriminant (pFisher <0.050 with Confidence Level of 95%) for hypotheses no. 1 through 7 respectively. Conclusion and Discussion: Results for the discriminant features identified from hypotheses no. 1 and 2 can be used to construct a predictive model with a higher performance that benefits from the zone-based information (PZ and CG) as a-priori knowledge. As most of the discriminant features in tests no. 1 to 7 are primarily entropy-based, this suggests that potential feature-based biomarkers of DILs in PCa patients are more associated with spatial-locality, and frequency-based characteristics of MR images. The high value of the OMPD for test no. 7, strongly supports the use of the two MR modalities for increasing the information gain in perfecting predictive models for detection of DILs in PCa studies. The results of this pilot study, albeit subject to confirmation in a larger patient population, suggest a potential role for the use of zone-based radiomics information in models developed for detection of DILs in PCa patients. Citation Format: Hassan Bagher-Ebadian, Branislava Janic, Chang Liu, Milan V. Pantelic, David Hearshen, Mohamed Elshaikh, Benjamin Movsas, Indrin J. Chetty, Ning Wen. Examination of zone-based radiomic features for characterization of dominant intraprostatic lesions using MR multi-modal information [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4539.

Abstract Purpose: This pilot study investigates a set of radiomics features extracted from fast relaxation fast spin echo (FRFSE) T2 pulse sequences for normal tissue and Dominant Intraprostatic Lesions (DILs) in twenty prostate cancer patients. Material and Methods: Twenty patients with prostate cancer were studied. All patients had axial FSRFSE T2 scans using a 3 Tesla scanner. A radiologist interpreted MR examinations, and contoured the suspicious DIL and the contralateral section of the prostate gland (normal) on the T2 weighted MR images. Patients underwent a 14-core transrectal Ultrasound Guided Biopsy and localization of positive cores, Gleason score and clinical tumor stage were recorded. 167 radiomics features were extracted from normal and DIL zones. These features were categorized into 8 different sets as following: Intensity Histogram Based (IHB), Gray Level Run Length (GLRL), Law’s Textural Information (LAWS), Discrete Orthonormal Stockwell Transform (DOST), Local Binary Pattern (LBP), Two Dimensional Wavelet Transform (2DWT), Two Dimensional Gabor Filter (2DGF), and Gray Level Co-Occurrence Matrix (GLCM) with 8, 7, 18, 18, 6, 48, 40, and 22 features in each category respectively. A Welch’s test and the Fisher method were used to test for significant differences among the 167 radiomics features and their subcategories. For all patients, correlation coefficients between the extracted features in the normal and DIL zones were also calculated. Results: According to the Fisher combined p-values, among the eight categories of radiomics features, only 5 feature categories showed a significant difference (IHB, GLRL, DOST, LBPF and GLCM with pFisher= 2.0x10-6, 0.02, 12 x10-4, 3.7 x10-3, and 1.5 x10-6 respectively). Among all 167 features, only 7 showed a significant difference (D=100x[DIL/NP-1]) and small correlation between normal and DIL zones: IHB-Skewness (r=0.19, p=0.03, and D=50.3%), GLCM-Contrast (r=0.12, p=0.03, and D=-67.5%), GLCM-Dissimilarity (r=0.12, p=0.01, and D=-67.5%), GLCM-Entropy (r=0.07, p=0.01, and D=-67.1%), GLCM-Difference-Variance (r=0.12, p=0.01, and D=-67.1%), GLCM-Difference-Entropy (r=0.10, p=0.01, and D=-60.4%), and GLCM-Information-Measure-of-Correlation (r=0.25, p=0.01, and D=-65.1%). Conclusion and Discussion: This pilot study demonstrates the feasibility of using radiomics features from MR images to characterize DILs in prostate cancer patients. Among 167 radiomics features extracted from axial MR T2 FRFSE, 7 features were shown to be potentially significant for distinguishing normal tissue from DILs. This research supports an integrated decision making system, combining clinical factors and radiomics features extracted from MR images, for increasing the DIL detection performance in prostate cancer studies. Citation Format: Hassan Bagher-Ebadian, Janic Branislava, Chang Liu, Milan Pantelic, David Hearshen, Indrin Chetty, Mohamed Elshaikh, Benjamin Movsas, Ning Wen. Examination and evaluation of MR radiomics features for characterization of dominant intraprostatic lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 555. doi:10.1158/1538-7445.AM2017-555

Systemic lupus erythematosus ( SLE ) is an autoimmune disease with a high prevalence of hypertension. NZBWF 1 ( SLE ‐Hyp) mice develop hypertension that can be prevented by modulating T cells. The peptide N ‐acetyl‐seryl‐aspartyl‐lysyl‐proline (Ac‐ SDKP ) decreases renal damage and improves renal function in a model of SLE without hypertension ( MRL / lpr ). However, it is not known whether Ac‐ SDKP prevents hypertension in NZBWF 1 mice. We hypothesized that in SLE ‐Hyp, Ac‐ SDKP prevents hypertension and renal damage by modulating T cells. Animals were divided into four groups: (1) control + vehicle, (2) control + Ac‐ SDKP , (3) SLE + vehicle, and (4) SLE + Ac‐ SDKP . Systolic blood pressure ( SBP ), albuminuria, renal fibrosis, and T‐cell phenotype were analyzed. SBP was higher in SLE compared to control mice and was not decreased by Ac‐ SDKP treatment. Half of SLE mice developed an acute and severe form of hypertension accompanied by albuminuria followed by death. Ac‐ SDKP delayed development of severe hypertension, albuminuria, and early mortality, but this delay did not reach statistical significance. Ac‐ SDKP prevented glomerulosclerosis, but not interstitial fibrosis in SLE ‐Hyp mice. SLE ‐Hyp mice showed a decrease in helper and cytotoxic T cells as well as an increase in double negative lymphocytes and T helper 17 cells, but these cells were unaffected by Ac‐ SDKP . In conclusion, Ac‐ SDKP prevents kidney damage, without affecting blood pressure in an SLE animal model. However, during the acute relapse of SLE , Ac‐ SDKP might also delay the manifestation of an acute and severe form of hypertension leading to early mortality. Ac‐ SDKP is a potential tool to treat renal damage in SLE ‐Hyp mice.

A hallmark of the tumor microenvironment in malignant tumor is extracellular acidosis, which can be exploited for targeted delivery of drugs and imaging agents. A pH sensitive paramagnetic nanoaparticle (NP) is developed by incorporating GdDOTA-4AmP MRI contrast agent and pHLIP (pH Low Insertion Peptide) into the surface of a G5 PAMAM dendrimer. pHLIP showed pH selective insertion and folding into cell membranes, but only in acidic conditions. We demonstrated that pHLIP-conjugated Gd-44-G5 paramagnetic nanoparticle binds and fuses with cellular membrane at low pH, but not at normal physiological pH, and that it promotes cellular uptake. Intracellular trafficking of NPs showed endosomal/lysosomal path ways.

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a naturally occurring tetrapeptide that prevents inflammation and fibrosis in hypertension and other cardiovascular diseases. We previously showed that, in angiotensin II-induced hypertension, Ac-SDKP decreased the activation of nuclear transcription factor NF-κB, whereas, in experimental autoimmune myocarditis and hypertension animal models, it also reduced the expression of endothelial leukocyte adhesion molecule ICAM-1. However, the mechanisms by which Ac-SDKP downregulated ICAM-1 expression are still unclear. TNF-α is a proinflammatory cytokine that induces ICAM-1 expression in various cell types via TNF receptor 1 and activation of the classical NF-κB pathway. We hypothesized that in endothelial cells Ac-SDKP suppresses TNF-α-induced ICAM-1 expression by decreasing IKK phosphorylation that as a consequence leads to a decrease of IκB phosphorylation and NF-κB activation. To test this hypothesis, human coronary artery endothelial cells were treated with Ac-SDKP and then stimulated with TNF-α. We found that TNF-α-induced ICAM-1 expression was significantly decreased by Ac-SDKP in a dose-dependent manner. Ac-SDKP also decreased TNF-α-induced NF-κB translocation from cytosol to nucleus, as assessed by electrophoretic mobility shift assay, which correlated with a decrease in IκB phosphorylation. In addition, we found that Ac-SDKP decreased TNF-α-induced IKK phosphorylation and IKK-β expression. However, Ac-SDKP had no effect on TNF-α-induced phosphorylation of p38 MAP kinase or ERK. Thus we conclude that Ac-SDKP inhibition of TNF-α activation of canonical, i.e., IKK-β-dependent, NF-κB pathway and subsequent decrease in ICAM-1 expression is achieved via inhibition of IKK-β.

The role of neutrophils in heart failure has been extensively analyzed and it has been shown that they are the first cells that massively invade the myocardium after myocardial infarction (MI). N‐Acetyl‐Seryl‐Aspartyl‐Lysyl‐Proline (Ac‐SDKP) is a natural tetrapeptide that is released from its precursor thymosin β4 (TB4). Ac‐SDKP and TB4 are shown to have beneficial effect in post MI cardiac healing by decreasing cardiac rupture and mortality in mice. Therefore we explored whether neutrophils play a role in the mechanism of AcSDKP and TB4 cardiac protection. We hypothesized that Ac‐SDKP and TB4 contribute to heart healing in MI by inhibiting neutrophil chemotaxis.To test neutrophil chemotaxis we performed transwell chemotaxis assay using neutrophil differentiated HL‐60 cells. We measured the migration of a) vehicle, b) Ac‐SDKP (10, 100 nM), or c) TB4 (20 nM) pre‐treated cells towards to a chemotactic bacterial formylated tripeptide N‐formyl‐met‐leu‐phe (fMLP) at 1nM. We also measured the effect of Ac‐SDKP on neutrophil infiltration post‐MI in mice. Ac‐SKDP or vehicle were infused subcutaneously using osmotic minipumps at a dose of 1.6 mg/kg/day. Two days after minipump implantation, MI was induced by permanent ligation of the left descending coronary artery. Neutrophil infiltration was measured at 24 hrs post‐MI by immunohistochemistry.At a dose of 100 nM Ac‐SDKP reduced fMLP‐induced neutrophil chemotaxis by 20 %. However, at 10 nM Ac‐SDKP failed to inhibit neutrophil chemotaxis. TB4 at 20 nM completely inhibited neutrophil migration. In vivo, Ac‐SDKP decreased neutrophil cardiac infiltration (veh: 44 vs Ac‐SDKP: 13 cells/mm2; p < 0.05).We conclude that TB4 is a potent inhibitor of neutrophil chemotaxis, while Ac‐SDKP exerts mild inhibitory effects. Inhibition of neutrophil chemotaxis could explain some of the beneficial effects of Ac‐SDKP and TB4 in post MI cardiac inflammation and fibrosis.Support or Funding InformationSupported by NIH HL028982 and AHA 13PRE17100074