Necrotic Regions Research Articles

Breast cancer proliferation and deterioration-associated metabolic heterogeneity changes induced by exposure of bisphenol S, a widespread replacement of bisphenol A

Exposure to bisphenol A (BPA) is considered to be associated with the increased incidence of breast cancer. As a widespread replacement of BPA, the effect of bisphenol S (BPS) on breast tumor programming has not been studied. We reported that BPS exposure significantly promoted proliferation and deterioration of breast tumor by nonmonotonic dose response. The mechanisms were investigated by molecular biology and mass spectrometry-based lipidomics, proteomics and imaging. BPS exposure induced the spatially intratumor heterogeneity of morphology-driven lipids and proteins. The more significant proliferation resulted from BPS-10 (10 μg/kg body weight /day) exposure was evidenced by the variations of spatial distribution of lipids related to ceramide-sphingomyelin signaling pathway, proteins related to chromosomal stability and cell proliferation in central necrotic regions of breast tumor. In contrast, the BPS-100 exposure obviously accelerated deterioration of breast tumor by the variations of spatial distribution of proteins that were associated with the stability of nucleic acid structure in peripheral neoplastic regions. Accordingly, dysregulation of metabolism and protein function as well as DNA methylation and hypoxic tumor microenvironment could be applied to predict the possibility of tumorigenesis, proliferation and metastasis that might be caused by other bisphenol analogs.

Extraosseous Ewing's Sarcoma: Pictorial Review of Imaging Findings, Differential Diagnosis, and Pathologic Correlation.

Extraosseous Ewing’s sarcoma (EES), first described in 1969, is a malignant mesenchymal tumor just like its intraosseous counterpart. Although Ewing’s sarcomas are common bone tumors in young children, EESs are rarer and more commonly found in older children/adults, often carrying a poorer prognosis. We discuss the multimodality imaging features of EES and the differential diagnosis of an aggressive appearing mass in proximity to skeletal structures, with pathologic correlates. This review highlights the need to recognize the variability of radiologic findings in EES such as the presence of hemorrhage, rich vascularity, and cystic or necrotic regions and its imaging similarity to other neoplasms that are closely related pathologically.

Screening of dual chemo-photothermal cellular nanotherapies in organotypic breast cancer 3D spheroids

Living therapeutics approaches that exploit mesenchymal stem cells (MSCs) as nanomedicine carriers are highly attractive due to MSCs native tropism toward the 3D tumor microenvironment. However, a streamlined pre-clinical evaluation of nano-in-cell anti-cancer therapies remains limited by the lack of in vitro testing platforms for screening MSCs-3D microtumor interactions. Herein we generated dense breast cancer mono and heterotypic 3D micro-spheroids for evaluating MSCs-solid tumors interactions and screen advanced nano-in-MSCs therapies. Breast cancer monotypic and heterotypic models comprising cancer cells and cancer associated fibroblasts (CAFs) were self-assembled under controlled conditions using the liquid overlay technique. The resulting microtumors exhibited high compactness, reproducible morphology and necrotic regions, similarly to native solid tumors. For evaluating tumoritropic therapies in organotypic tumor-stroma 3D models, theranostic polydopamine nanoparticles loaded with indocyanine green-doxorubicin combinations (PDA-ICG-DOX) were synthesized and administered to human bone-marrow derived MSCs (hBM-MSCs). The dual-loaded PDA nano-platforms were efficiently internalized, exhibited highly efficient NIR-light responsivity and assured MSCs viability up to 3 days. The administration of PDA-ICG-DOX nano-in-MSC tumoritropic units to microtumor models was performed in ultra-low adhesion surfaces for simulating in vitro the stem cell-tumor interactions observed in the in vivo scenario. Bioimaging analysis revealed hBM-MSCs adhesion to 3D cancer cells mass and MSCs-chemo-photothermal nanotherapeutics exhibited higher anti-tumor potential when compared to their standalone chemotherapy treated 3D tumor counterparts. Overall, the proposed methodology is suitable for evaluating MSCs-microtumors individualized interactions and enables a rapid high-throughput screening of tumoritropic therapies bioperformance.

Numerical analysis of non-Fourier thermal response of lung tissue based on experimental data with application in laser therapy

Background and objectiveThe thermal therapy is a minimally invasive technique used as an alternative approach to conventional cancer treatments. There is an increasing concern about the accuracy of the thermal simulation during the process of tumor ablation. This study is aimed at investigating the effect of finite speed of heat propagation in the biological lung tissue, experimentally and numerically. MethodsIn the experimental study, a boundary heat flux is applied to the lung tissue specimens and the temperature variation is measured during a transient heat transfer procedure. In the numerical study, a code is developed based on the finite volume method to solve the classical bio-heat transfer, the Cattaneo and Vernotte, and the Dual-phase-lag (DPL) equations. The thermal response of tissue during the experiments is compared with the predictions of the three heat transfer models. ResultsIt is found that the trend of temperature variation by the DPL model resembles the experimental results. The experimental observation in parallel with the numerical results reveals that the accumulated thermal energy diffuses to the surrounding tissue with a slower rate in comparison with the conventional bio-heat transfer model. The DPL model is implemented to study the temperature elevation in the laser irradiation to lung tissue in the presence of gold nanoparticles (GNPs). It is concluded that the extent of the necrotic tumoral region and the area of the damaged healthy tissue are reduced, when the non-Fourier heat transfer is taken into account. ConclusionsResults show that considering the phase lags is crucial in planning for an effective thermal treatment, in which the cancerous tissue is ablated and the surrounding tissues are preserved from irreversible thermal damage.

Saturation transfer properties of tumour xenografts derived from prostate cancer cell lines 22Rv1 and DU145

Histopathology is currently the most reliable tool in assessing the aggressiveness and prognosis of solid tumours. However, developing non-invasive modalities for tumour evaluation remains crucial due to the side effects and complications caused by biopsy procedures. In this study, saturation transfer MRI was used to investigate the microstructural and metabolic properties of tumour xenografts in mice derived from the prostate cancer cell lines 22Rv1 and DU145, which express different aggressiveness. The magnetization transfer (MT) and chemical exchange saturation transfer (CEST) effects, which are associated with the microstructural and metabolic properties in biological tissue, respectively, were analyzed quantitatively and compared amongst different tumour types and regions. Histopathological staining was performed as a reference. Higher cellular density and metabolism expressed in more aggressive tumours (22Rv1) were associated with larger MT and CEST effects. High collagen content in the necrotic regions might explain their higher MT effects compared to tumour regions.

T1-weighted and T2-weighted Subtraction MR Images for Glioma Visualization and Grading.

To evaluate the performance of multiparametric MR images in differentiation of different regions of the gross tumor area and for assessment of glioma grade. Forty-six glioma subjects (18 grade II, 11 grade III, and 17 grade IV) underwent a comprehensive MR and spectroscopic imaging procedure. Maps were generated by subtraction of T1-weighted images from contrast-enhanced T1-weighted images (ΔT1 map) and T1-weighted images from T2-weighted images (ΔT2 map). Regions of interest (ROIs) were positioned in normal-appearing white matter (NAWM), enhancing tumor, hyperintense T2, necrotic region, and immediate and distal peritumoral regions (IPR and DPR). Relative signal contrast was estimated as difference between mean intensities in ROIs and NAWM. Classification using support vector machines was applied to all image series to determine the efficacy of regional contrast measures for differentiation of low- and high-grade lesions and grade III and IV lesions. ΔT1 and ΔT2 maps offered higher contrast as compared to other parametric maps in differentiating enhancing tumor and edematous regions, respectively, and provided the highest classification accuracy for differentiating low- and high-grade tumors, of 91% and 90.4%. Choline/N-acetylaspartate maps provided significant contrast for delineating IPR and DPR. For differentiating high-grade gliomas, ΔT2 and ΔT1 maps provided a mean accuracy of 90.9% and 88.2%, which was lower than that obtained using cerebral blood volume (93.7%) and choline/creatine (93.3%) maps. This study showed that subtraction maps provided significant contrast in differentiating several regions of the gross tumor area and are of benefit for accurate tumor grading.

Stem cell therapy for osteonecrosis of femoral head: Opportunities and challenges.

Osteonecrosis of the femoral head (ONFH) is a progressive disease with a complex etiology and unclear pathogenesis, resulting in severe hip pain and dysfunction mainly observed in young patients. Although total hip arthroplasty (THA) is the most effective treatment for patients with ONFH in the terminal stage, the results of THA in young patients or active populations are often not favorable, with some complications related to the prosthesis. With the development of biotechnology, an increasing number of studies pay attention to use of stem cells for the treatment of ONFH. Stem cells are characterized by the ability to self-renew and differentiate into multiple cell types, including differentiation into osteoblasts and endothelial cells to mediate bone repair and angiogenesis. Furthermore, stem cells can offer growth factors to promote blood supply in the necrotic regions by paracrine effects. Therefore, stem cell therapy has become one of the hip-preserving alternatives for ONFH. This review summarized the current trends in stem cell therapy for ONFH, from clinical applications to related basic research, and showed that an increasing number of studies have confirmed the effectiveness of stem cell therapy in ONFH. However, many unsolved problems and challenges in practical applications of stem cell therapy still exist, such as patient selection, standardized procedures, safety assessment, and the fate of transplanted cells in the body. Additional studies are required to find ideal cell sources, appropriate transplantation methods, and the optimal number of cells for transplantation.

Studies on Leaf Blight Disease of Sissoo (Dalbergia sissoo Roxb.) in Bangladesh

The outbreak of leaf blight disease in sissoo (Dalbergia sissoo Roxb.) was detected in different plantation areas of Sylhet, Bangladesh, during surveys conducted in November 2019. Isolates were consistently recovered from the necrotic region of the blight infected leaves. Isolates grown on potato-dextrose-agar (PDA) showed greyish-white cottony mycelia with a concentric zone of orange conidial masses. Average conidia length and width ranging from 13.5-17.7 µm and 3.5-5.3 µm, respectively. In the pathogenicity test, the pathogen was able to cause infection on detached healthy leaves and spots developed were similar to those observed on the leaves affected in nature. However, the pathogen produced disease symptoms in wounded leaves but did not produce any symptoms on the non-wound ones. Findings also suggested that the pathogen was equally virulent to three-leaf grades (young, middle and mature-aged). Based on the above morphological features, the pathogen was identified as Colletotrichum sp. Molecular identification is needed to determine the pathogen up to species. The observation of the pathogen causing leaf blight disease of sissoo in Bangladesh has severe implications regarding the management of plantations and nurseries. More surveys are needed to determine the distribution and extent of damage caused by the pathogen in other regions.

Role of Myeloid-Derived Suppressor Cells in High-Dose-Irradiated TRAMP-C1 Tumors: A Therapeutic Target and an Index for Assessing Tumor Microenvironment

To investigate the temporal and spatial infiltration of TRAMP-C1 tumors by myeloid-derived suppressor cells (MDSCs) after high-dose radiation therapy (RT), and to explore their effect on tumor growth. TRAMP-C1 intramuscularly tumors were irradiated with a single dose of 8 Gy or 25 Gy. The dynamics of infiltrated MDSCs and their intratumoral spatial distribution were assessed by immunohistochemistry and flow cytometry. Cytokine levels in the blood and tumor were analyzed by multiplex immunoassay. Mice were injected with anti-Gr-1 antibody to determine whether MDSCs affect tumor growth after RT. CD11b+Gr-1+ MDSCs infiltrated TRAMP-C1 tumors irradiated with 25 Gy, but not 8 Gy, within 4 hours and recruitment persisted for at least 2 weeks. Both CD11b+Ly6G+Ly6C+ polymorphonuclear-MDSCs (PMN-MDSCs) and CD11b+Ly6G-Ly6Chi monocytic-MDSCs (M-MDSCs) were involved. Tumor RT also increased the representation of both MDSC subpopulations in the spleen and peripheral blood. Levels of multiple cytokines were increased in the tumors at 2 weeks, including GM-CSF, G-CSF, CCL-3, CCL-5, CXCL-5, IL-6, IL-17α, and VEGF-a; while G-CSF, IL-6, and TNF-α levels increased in the blood. PMN-MDSCs aggregated in the central necrotic region of the irradiated tumors over time, where they were associated with avascular hypoxia (CD31-PIMO+). MDSCs expressed the proangiogenic factor, matrix metalloproteinase-9, and, within the necrotic area, high levels of arginase-1 and indoleamine 2,3-dioxygenase. Depletion of PMN-MDSCs by Gr-1 antibody increased the efficacy of high-dose RT. PMN-MDSCs infiltrate TRAMP-C1 tumors after high-dose RT. Their spatial distribution suggests they are involved in the evolution of an intratumoral state of necrosis associated with avascular hypoxia, and their phenotype is consistent with them being immunosuppressive. They appear to promote tumor growth after RT, making them a prime therapeutic target for therapeutic intervention. Assessment of MDSCs and cytokine levels in blood could be an index of the need for such an intervention.

STEM-08. PLATELETS DRIVES GLIOBLASTOMA ONCOGENESIS BY ENHANCING THE GLIOMA STEM CELL PHENOTYPE

Abstract Glioblastoma (GBM) is recognized as one of the deadliest forms of cancer, despite aggressive therapy consisting of maximal surgical resection followed by concurrent radiation and chemotherapy, the median survival remains ~15 months. Glioma stem cells (GSCs) possess potent tumor-initiating properties and comprise a cellular hierarchy that is responsible for treatment resistance and progression. Specifically targeting GSCs has been considered a promising therapeutic approach, however no clear method has been identified. Histologically, it is known that GSCs are found in perivascular and pseudsopalisading regions of GBM. Similarly, platelet aggregates are often found in pseudsopalisading necrotic regions, suggesting a potential interaction between platelets and GSCs due to their spatial locations. High platelet counts have been associated with poor clinical outcome in many cancers including ovarian and endometrial cancer. While platelets are known to affect progression of other tumors, mechanisms by which platelets influence GBM oncogenesis are unknown. Our work aimed to understand the crosstalk between GSCs and platelets within GBM solid tumors that work to enhance disease progression and treatment resistance. Our clinical studies suggest elevated platelet counts positively correlate with tumor growth and negatively correlate to overall patient survival. We found platelets and GSC co-localization in GBM solid tissue; platelet exposure to GSCs results in increased proliferation of GSCs specifically, by increasing the self-renewing capacity of GSCs in a dose dependent manner, and resulted in an increased “Stem-like” transcriptional pattern. Consequently, inhibiting the GSC-platelet interaction results in a decrease in GSC renewal and stemness. These results introduce a novel interaction between GSCs and platelets and elucidate a novel therapeutic approach specifically targeting GSCs by disrupting the GSC-platelet interaction.

STEM-04. PLATELETS DRIVE GLIOBLASTOMA ONCOGENESIS BY ENHANCING THE GLIOMA STEM CELL PHENOTYPE

Abstract Glioblastoma (GBM) is recognized as one of the deadliest forms of cancer, despite aggressive therapy consisting of maximal surgical resection followed by concurrent radiation and chemotherapy, the median survival remains ~12 months. Glioma stem cells (GSCs) possess potent tumor-initiating properties and comprise a cellular hierarchy that is responsible for treatment resistance and progression. Specifically targeting GSCs has been considered a promising therapeutic approach, however no clear method has been identified. Histologically, it is known that GSCs are found in perivascular and pseudsopalisading regions of GBM. Similarly, platelet aggregates are often found in pseudsopalisading necrotic regions, suggesting a potential interaction between platelets and GSCs due to their spatial locations. High platelet counts have been associated with poor clinical outcome in many cancers including ovarian and endometrial cancer. While platelets are known to affect progression of other tumors, mechanisms by which platelets influence GBM oncogenesis are unknown. Our work aimed to understand the crosstalk between GSCs and platelets within GBM solid tumors that work to enhance disease progression and treatment resistance. Our clinical studies suggest elevated platelet counts positively correlate with tumor growth and negatively correlate to overall patient survival. We found platelets and GSC co-localization in GBM solid tissue; platelet exposure to GSCs results in increased proliferation of GSCs specifically, by increasing the self-renewing capacity of GSCs in a dose dependent manner, and resulted in an increased “Stem-like” transcriptional pattern. Inhibiting the GSC-platelet interaction results in a decrease in GSC renewal and stemness. These results introduce a novel interaction between GSCs and platelets and elucidate a novel therapeutic approach specifically targeting GSCs by disrupting the GSC-platelet interaction.

On-Tissue Spatially-Resolved Glycoproteomics Guided by N-Glycan Imaging Reveal Global Dysregulation of Canine Glioma Glycoproteomic Landscape

We identify N-linked glycoproteins and deduce their spatial localization using spatially-resolved glycoproteomic guided by MALDI mass spectrometry N-glycan imaging on formalin-fixed paraffin embedded (FFPE) canine high-grade glioma tissues. The glycan HexNAc4-Hex5-NeuAc2 was found to be predominantly expressed in necrotic regions, whereas high mannose HexNAc2-Hex5 was predominantly found in benign regions. To determine the underlying sialo-glycoprotein, various regions in adjacent tissue sections were analysed. Haptoglobin is the protein associated with HexNAc4-Hex5-NeuAc2. Additionally, we identified several high-mannose (Hex2-HexNAc5) glycopeptides enriched in benign regions. This is the first report that directly links glycan imaging with intact glycopeptide identification. Over 400 N-glycosylated, O-GalNAcylated, O mannosylated, and S- and O- GlcNAcylated glycopeptides from over 30 proteins were identified. While N-glycosylation and O- mannosylation were similar between benign and tumor/necrotic sections, S- and O-GlcNAc glycopeptides were significantly decreased in tumor/necrotic sections, whereas sialylated O-GalNAc glycopeptides were significantly upregulated.

Can B Washout Rate be a Biomarker of Tumor Viability in Charged Particle Therapy? A Rat In-beam PET Study

Positron emission tomography (PET) is a practical tool for range verification in charged particle therapy. We aimed to apply in-beam PET imaging for tracking the evolution of irradiated particles in living tissue. The quantitative biological washout rate of the positron emitters has a potential usefulness as a diagnostic index that provides a unique opportunity to probe the status of tumor viability, but the modeling for this has not been established. Therefore, we measured biological washout rates of tumor-bearing nude rat and performed kinetic analysis on dynamic PET data to explore the biological washout mechanism. C6 rat glioma cells were implanted into right shoulder subcutaneous regions of six nude rats. Tumors volume reached to about 3 cm3 on day 14 after implantation. Tumor tissue (right shoulder) and normal tissue (left shoulder) were irradiated by 11C radioactive ion beam. Time activity curves (TACs) of the irradiated field were acquired by our original PET prototype. The biological washout rate was quantified based on the two-washout model assuming medium decay rate (k2m) and slow decay rate (k2s). In this model, k2m is speculated as the component which spread out via interstitial fluid, and k2s is speculated as the trapping component by the stable molecules in the tissue. After irradiation and PET dynamic scan, pathological morphology of tumor tissue (Hematoxylin & Eosin staining) was observed under microscope. The two-washout model fitted the experimental TACs well for five of six nude-rats irradiation studies. The observed k2m of tumor irradiation was 0.31+/-0.04 min-1, which was consistent with the normal tissue irradiation. While k2s of tumor irradiation was faster (0.017 +/-0.002 min-1) than that of normal tissue irradiation (0.006 +/-0.002 min-1). These results suggested that the component, which was trapped in normal tissue, diffused out more smoothly from tumor tissue due to vascular hyperpermeability which characterizes pathological tumor-angiogenesis. For one of six nude rats, only slow washout component (k2s = 0.014 +/-0.003 min-1) was observed in tumor irradiation study. Necrosis in tumor was observed in pathological tissue sample of this nude rat. It is also suggested that the decrease of washout rate was caused by the decrease of blood flow in the necrotic region. We measured the biological washout rate of irradiated 11C ion beam in tumor tissue and normal tissue. The physiological difference of tissue condition reflected the biological washout rates. This effect may be used as a clinical diagnostic index to monitor the dose response of the tumors in individual patients as well as the observation of the functional metabolism of tumor.

The Impact of the Electric Conductivity of Tissues on the Electric Field Intensity and Power Density during TTFields Therapy for Glioblastoma

Tumor Treating Fields (TTFields) is an FDA-approved technique used in the treatment of glioblastoma (GBM). It consists in inducing an electric field (EF) in the head in two perpendicular directions alternately using a specific device that operates at 200 kHz. Post-hoc analyses of the EF-14 clinical trial results showed that an intensity of at least 1.06 V/cm and a minimum power density (PD) of 1.15 mW/cm3 at the tumor bed can significantly affect the mitotic process of tumoral cells. Given that it is not possible to experimentally measure these quantities, computational modelling is widely used to predict treatment effectiveness. However, the uncertainties in the electric conductivity (σ) of biological tissues affect the accuracy of these predictions. The goal of this work is to investigate for which tissues the uncertainty associated with σ values leads to the highest variations in these metrics and quantify their impact. A realistic head model divided into scalp, skull, CSF and brain was used in this study. A virtual lesion was added at the right hemisphere near the ventricle. This lesion intended to represent a GBM tumor and it consisted in a necrotic region surrounded by an enhancing part. Two pairs of transducer arrays were placed on the scalp. The first was placed over the supraorbital region and at the back of the head (Anterior-Posterior or AP configuration) and the other over the left and right temporal and parietal regions (Left-Right or LR configuration). To perform the sensitivity analysis of the most significant electric parameters, an extensive literature review was performed to obtain a range of σ values for each tissue represented. The average values of the EF intensity and PD were analyzed in a volume of interest (VOI) containing the enhancing tumor plus a 3-mm-thick proximal boundary surrounding it. Given that this array layout was not optimized for this specific tumor location the AP configuration did not produce a therapeutic EF at the tumor bed and thus just the results for the LR configuration were analyzed. The highest variation in the EF intensity in the VOI was seen when changing the electric conductivity of the white matter. Decreasing it led to a maximum of 1.82 V/cm, whereas increasing it to the maximum value reported in the literature led to a minimum EF intensity of 1.14 V/cm. On the other hand, the minimum value for the PD occurred when skull’s conductivity was reduced, 1.16 mW/cm3, while the maximum was obtained when scalp’s conductivity was diminished, 2.69 mW/cm3. These results indicate that the uncertainty associated with the electric parameters of tissues might lead to significant variations in the metrics used to evaluate TTFields efficacy. Experimental measurements are necessary to determine these values with precision and thus to increase the accuracy of data obtained through computational simulations.

The Role of Myeloid-derived Suppressor Cells in High-dose Irradiated TRAMP-C1 tumors: A Therapeutic Target and an Index for Assessing Tumor Microenvironment

To investigate the chronological and spatial responses of myeloid-derived suppressor cells (MDSCs) to high-dose irradiation in TRAMP-C1 tumor and explore their potential role in tumor re-growth after irradiation. Murine prostate TRAMP-C1 tumors were irradiated with a single dose of 25Gy (SD-RT). Using immunohistochemical staining (IHC) and flow cytometry to verify the spatial distribution and dynamic mobilization of MDSCs. Blood and tumor lysate were collected for multiplex immunoassay. Furthermore, mice were injected Gr-1 antibody to verify the role of MDCSs in tumor re-growth after irradiation. Recruitment of Gr-1+ cells into irradiated tumors rapidly occurred in 4 hours and persisted up to 2 weeks. Flow cytometry showed that the main subpopulations of recruited MDSCs within the tumor are CD11b+Ly6G+Ly6C+ polymorphonuclear-MDSCs (PMN-MDSCs) and CD11b+Ly6G-Ly6Chi monocytic-MDSCs (M-MDSCs), not CD11b+Ly6G-Ly6C- tumor-associated macrophages (TAMs). The percentage of PMN-MDSCs and M-MDSCs was also increased in peripheral blood after radiation therapy. At two weeks after irradiation, levels of multiple cytokines, including GM-CSF, G-CSF, CCL-3, CCL-5, CXCL-5, IL-6, IL-17α, and VEGF-a were increased in the irradiated tumors; and levels of G-CSF, IL-6 and TNF-α were increased in the blood. The PMN-MDSCs chronologically aggregated at the central necrotic region of chronic hypoxia. The infiltrated Ly6G + PMN-MDSCs dominantly possessed pro-angiogenic factor, matrix metalloproteinase-9 (MMP-9), and highly expressed arginase-1, an immunosuppressive molecule, at the necrotic area. The depletion of MDSCs by the Gr-1 antibody had no effects on sham-irradiated tumors, but significantly delayed the regrowth of irradiated tumor. PMN-MDSCs are actively involved in tumor responses to high-dose irradiation, and their spatial distribution and phenotype changes are associated with the evolution of chronic hypoxia in the irradiated tumors. PMN-MDSCs could become a therapeutic target. Checking the MDSCs component and cytokine levels in peripheral blood could be used as an index for assessing responses in the tumor microenvironment.

Machining-induced thermal damage in cortical bone: Necrosis and micro-mechanical integrity

In bone cutting, the tissue is exposed to necrosis due to temperature elevation, which can significantly influence postoperative results in orthopaedic surgeries. This damage is usually revealed through histological analysis to show the necrotic extent; however, this technique does not capture mechanical damage, which is essential for a full material integrity assessment. Here, with micro-mechanics, it is demonstrated that machining-induced damage in bone extends beyond the necrotic region. Drilling with different conditions was performed on ex-vivo bovine cortical bone, inducing different damage degrees. Micro-pillar compression tests were performed in the machined sub-surface to identify changes in properties and failure modes caused by drilling. It was revealed that at high cutting temperatures, the bone near the machined surface suffers from lower modulus (−42%), strength (−41%) and brittle behaviour, whereas the bulk bone remains undamaged with pristine properties and ductile behaviour. Histology was also performed to evaluate necrosis and, surprisingly, it was found that the brittle and weaker bone layer is more than three times larger when compared to the necrotic layer, clearly showing that the drilling thermo-mechanical effect could affect not only biologically, but also micro-mechanically. Consequently, these results reveal another kind of bone damage that has so far been neglected.

Numerical simulation of the effect of necrosis area in systemic delivery of magnetic nanoparticles in hyperthermia cancer treatment.

In a magnetic hyperthermia treatment, malignant cancerous cells are ablated by the heat production of magnetic nanoparticles (MNP) under an external magnetic field. This novel approach is a promising tool to eliminate the tumor cells by a higher temperature inside the tumor microenvironment. MNPs are needed inside the tumor microenvironment to increase the heat, and this could be possible with intravenous drug injection. However, tumors with necrosis regions are more resistant to drug penetration, and this can cause inadequate and non-homogeneous temperature distribution in the tumor. Hence, in this study, we used numerical methods to investigate the Spatio-temporal temperature field distribution in the necrotic tumor and its surrounding tissue. To this end, an intravenous bolus injection is used to simulate the effect of systemic drug delivery in tumors with necrosis region. Results show that the temperature field with the necrosis region with 10% of the tumor radius is more prone to higher temperature values. The hypoxia region is affected by the high temperature despite the necrosis region in the tumor. However, a broader necrosis region impedes drug penetration inside the inner layers of tumors, which leads to a lower heat generation by the MNPs. Results also demonstrate that only 15.5% of MNP concentration distributed to the necrosis with 50% of tumor radius, leading a temperature of 42∘C in the necrosis region, which is not sufficient for the tumor ablation. Therefore, the temperature distribution is dependant on the sizes of necrosis regions in tumors, and tumors with a larger necrotic region (over 20% of tumor radius) are challenging to treat with hyperthermia treatment. This study could help the future in vitro and in vivo studies of hyperthermia treatment in necrotic tumors.

Abstract CT251: Immunological and histopathological tumor responses to a novel neoadjuvant peptide vaccine targeting HSP70 and GPC3 antigens in patients with resectable HCC

Abstract Background: Even with curative resection, the recurrence rate of HCC remains high, and effective adjuvant therapies are not currently available. Our previous Phase I study with novel therapeutic peptides and immune adjuvants demonstrated safety, antigen-specific CTL induction in PBMC, and a sign of efficacy (ASCO 2017 Abstract # 3086). We thus started a Phase I study of the same vaccination therapy as a perioperative immunotherapy setting in patients with resectable HCC (jRCTs061180058). Methods: Two mg each of HLA-A*24:02, 02:01, or 02:06-restricted HSP70- and GPC3-derived peptides, in combination with hLAG-3Ig (1.0 mg) + poly-IC:LC (1.4 mg) were injected intradermally at four sites of the inguinal and axillary regions every week for 6 weeks before surgery. Patients subsequently received 10 injections of adjuvant immunotherapy over 4 months. Surgical specimens and PBMCs were analyzed by mass cytometry (CyTOF), using 66 antibodies to monitor T-cell exhaustion, T-cell activation, and effector Treg induction. Tumor specimens were also subjected to HE staining and immunohistochemical staining for CD3, CD8, PD1, HSP70, and GPC3. Results: Nine patients were treated with preoperative vaccination, and resected surgical specimens and PBMC were examined. Pathohistological analysis revealed three response types: “hot cellular immunity”, “intermediate fumoral immunity”, and “cold response”. In three patients, massive infiltration of CD8+ and PD1+ T cells accompanied necrotic and fibrous regions, in which the targeted tumor antigens HSP70 and GPC3 were highly expressed, representing “hot cellular immunity”. Regarding “intermediate”, three patients showed necrotic regions with infiltrating macrophages. HSP70 and/or GPC3 were also highly expressed, but little infiltration of lymphocytes was evident. In the remaining three patients, “cold response” manifested as tumor comprising a pseudo-glandular pattern with no infiltration of T cells or macrophages. Two of these three patients showed no expression of targeted tumor antigens. CyTOF analysis of the tumors also revealed the percentages of CD3+ cells among live cells and of PD1+ cells among CD8+ cells were extremely low in cold response. However, this trend was not observed in PBMCs, suggesting the critical importance of TIL analysis. Conclusions: This novel therapeutic peptide and immune adjuvant combination induced sustained immune cell infiltration into tumor microenvironments, especially in those presenting target tumor-associated antigens with a non-pseudo-glandular type. Our novel immunotherapy may convert cold tumors into hot tumors containing PD1+ lymphocytes. Combining this novel strategy with PD (L) 1 antibody may be warranted. Citation Format: Masao Nakajima, Shoichi Hazama, Koji Tamada, Keiko Udaka, Shun Doi, Michiie Sakamoto, Akira Saito, Shiro Akinaga, Hiroto Matsui, Yoshitaro Shindo, Satoshi Matsukuma, Yukio Tokumitsu, Shinobu Tomochika, Michihisa Iida, Shin Yoshida, Ryouichi Tsunedomi, Yuki Nakagami, Nobuaki Suzuki, Shigeru Takeda, Hiroaki Nagano. Immunological and histopathological tumor responses to a novel neoadjuvant peptide vaccine targeting HSP70 and GPC3 antigens in patients with resectable HCC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT251.

Abstract 3325: Intratumoral STING activation on artificially generated cutaneous metastasis of EML4-ALK lung cancer alters immunologic milieu of primary tumor

Abstract Introduction: Therapeutic monoclonal antibodies targeting immune checkpoints inhibitors (ICIs) are game changers in the treatment landscape of multiple solid tumors. However, response rate remains relatively low in most cases. Particularly, anti-PD-1 or PD-L1 therapies are not effective in EML4-ALK rearranged- and EGFR mutant-NSCLC patients due to their cold immune microenvironment. Recently, activation of stimulator of interferon genes (STING) has shown potential to enhance antitumor immunity through induction of various pro-inflammatory cytokines and chemokines. In this study, we attempted to induce inflamed tumor microenvironment by artificially generating skin metastasis using intratumoral STING activation in EML4-ALK transgenic mice. Methods: EML4-ALK transgenic mice were implanted with a new EML4-ALK tumor subcutaneously. The size of lung primary tumor and subcutaneous tumor were each measured by micro-MRI and calipers, respectively. The treatment groups were divided into the control group, tumor-implanted group, and intratumor STING activation group (skin). On day 14, subcutaneous tumor, primary tumor (lung), and systemic immune organs were collected for further analysis. Results: STING agonist promotes pIRF3 signaling and massive immune cell recruitment in skin region, the subcutaneous tumor was dramatically reduced with hyperinflammation. In addition, primary tumor (lung) was observed with increased T cell population in tumor with necrotic regions. IFN-gamma expression was increased in bronchoalveolar fluid in STING activation group compared to control and tumor only groups (P < 0.05). Tumor macrophages and NK cells were increased compared to control group (P < 0.05, P = ns, respectively). We considered that skin consists of cutaneous innate immune cells, especially, macrophage. Increased T cells and macrophages were observed in STING activated tumor within 6 hours. The in vitro assay proved that macrophages highly express chemokines (CCL5, CCL2, and CCL12) which is recruiting T cells and myeloid cells. This initial immune activation results in increased tumor-specific T cell, especially, memory helper T cell (CD3+CD4+CD44+) in spleen, LN and MLN. We tested anti-IFN-beta antibody for neutralization of STING activity in vivo. The IFN-gamma expression of lymphocytes in MLN was reduced but not significant. Conclusion: This study confirmed that STING activation in cold tumor alters inflamed-immunologic milieu of primary tumor via hyper cutaneous immune activity with pivotal roles of macrophage. Our findings provide a rationale for further clinical investigations. Keywords: STING, cutaneous metastasis, EML4-ALK Citation Format: Chun-Bong Synn, Ji Min Lee, SunMin Lim, Do Hee Kim, Bianca-Ioana Gheorghiu, Jae-Hwan Kim, Wongeun Lee, Sung-Eun Kim, Sungwon Park, Beung-Chul Ahn, Ha Ni Jo, Min Hee Hong, Hye Ryun Kim, Kyoung-Ho Pyo, Byung Chul Cho. Intratumoral STING activation on artificially generated cutaneous metastasis of EML4-ALK lung cancer alters immunologic milieu of primary tumor [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3325.

Abstract 6575: A novel AI-driven spatial genomics platform enables tumor enrichment and analysis of heterogeneity

Abstract Spatial genomics and digital pathology continue to advance at a rapid pace, generating large amounts of data. There is a critical unmet need for advanced tools to expedite spatial data generation, visualization, and analysis, to facilitate an improved understanding of health and disease, especially with the advancement of personalized cancer treatments. In this study we present a novel method for the extraction and analysis of genomic information from specific regions of interest (ROI) within a series of formalin-fixed paraffin embedded (FFPE) breast cancer adenocarcinoma tissue sections. An artificial intelligence-based tumor profiling algorithm first identified tumor, stroma, inflammatory, and necrotic regions within the tissue, and segmentation results were confirmed by a Board Certified Pathologist. Automated lysate extraction was then performed in the selected ROIs using ink-jet technology and novel ink chemistry. The process was performed directly on the tissue slide and the crude lysates generated were transferred to a standard collection tube for subsequent sequencing. DNA was extracted from the selected areas before being purified and analyzed using a multi-cancer panel. Library QC showed optimal coverage at an average of >95% uniformity. Sequencing results showed an average coverage of 95% at 250x across all amplicons of the panel. Variant calling was successfully performed. Sequencing data is presented to demonstrate the high coverage achieved using this workflow as well as the resulting tumor heterogeneity. This novel technology provides an efficient solution for the enrichment of tumor content with high sequencing coverage from FFPE tissue sections. Novel spatial data visualization and analysis techniques allowed a comprehensive assessment of tumor heterogeneity. Further studies on both DNA and RNA should be conducted to validate this approach for clinical molecular testing. Citation Format: Bidhan Chaudhuri, Katie Konigsfeld, Marisa Sanchez, Jason Smith, Casey Laris, Claire Weston, John Butler. A novel AI-driven spatial genomics platform enables tumor enrichment and analysis of heterogeneity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6575.