Areas Of Tumor Invasion Research Articles

Abstract 1509: Microglial dominance in glioma dissemination: Unraveling TAMs' impact and therapeutic implications through modulating cathepsin activity

Abstract Introduction: It is well known that tumor-associated macrophages/microglia (TAMs) play a vital role in brain tumor progression. Our previous studies suggested that the activated microglia could reduce the cytotoxic effect of chemotherapeutic drugs on glioma cells in a 2D/3D model. This study delves into TAMs within diverse brain TME regions, emphasizing the distinction of resident microglia from infiltrating macrophages in invasion areas and their role in glioma migration and invasion. Material and Method: We employed the murine orthotopic astrocytoma model (ALTS1C1), dissecting brain tumor tissues into normal, invasion, edge, and core regions. Immunofluorescence staining, utilizing TMEM119 and CD68 markers to identify resident microglia and infiltrating macrophages, respectively. The IHC results were analyzed with QuPath software and validated via flow cytometry. Microglia (BV2) and macrophage (RAW264.7) cell lines were directly co-cultured with ALTS1C1 in 2D and 3D migration/invasion models. Bulk RNA sequencing analysis unraveled the differences between BV2 and RAW264.7. Bone marrow-derived macrophages (BMDM) were used for the TAMs activation test, and the cathepsin inhibitor, E64, was applied to elucidate cathepsin's role in microglia within co-culture models. Results: The IHC results reveal more TMEM119+ microglia than CD68+ infiltrating macrophages in the tumor invasion area. On the other hand, there are more CD68+ infiltrating macrophages than TMEM119+ microglia in the tumor core regions, signifying a specific role of microglia in tumor invasion. Flow cytometry further supported that more CD11b+CD45low microglia prevalence in tumor surrounding tissues than in tumor core. ALTS1C1 astrocytoma cells, in the presence of microglia, exhibited more increased invasion and migration ability than the presence of macrophages in 2D/3D models. RNA sequencing highlighted M2 activation and cathepsin gene upregulation in microglia over macrophages. M2-polarized TAMs enhanced 2D migration, while cathepsin inhibition significantly decreased glioma's 3D invasion potential. Conclusion: This study identifies microglia as primary TAMs in brain tumor invasion areas and their roles in glioma cell survival and dissemination. Notable differences in activation states and the critical impact of cathepsin in microglia for their roles on brain tumor invasion and survival offer promising therapeutic avenues. Targeting cathepsin in microglia emerges as a novel glioma prevention and treatment strategy. These findings contribute valuable insights to enhance clinical interventions for glioma patients. Citation Format: Sheng-Yan Wu, Chi-Shiun Chiang. Microglial dominance in glioma dissemination: Unraveling TAMs' impact and therapeutic implications through modulating cathepsin activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1509.

Role of amide proton transfer imaging in maximizing tumor resection in malignant glioma: a possibility to take the place of 11C-methionine positron emission tomography.

Amide proton transfer (APT) imaging has been proposed as a technique to assess tumor metabolism. However, the relationship between APT imaging and other quantitative modalities including positron emission tomography (PET) has not been investigated in detail. This study aimed to evaluate the clinical usefulness of APT imaging in determining the metabolic status of malignant glioma and to compare findings with those from 11C-methionine (Met)-PET. This research analyzed APT imaging data from 20 consecutive patients with malignant glioma treated between January 2022 and July 2023. Patients underwent tumor resection and correlations between tumor activity and intensity of APT signal were investigated. We also compared 11C-Met-PET and APT imaging for the same regions of the perifocal tumor invasion area. Clear, diagnostic APT images were obtained from all 20 cases. Mean APT intensity (APTmean) was significantly higher in the glioblastoma (GBM), IDH wild type group (27.2 ± 12.8%) than in other gliomas (6.0 ± 4.7%; p < 0.001). The cut-off APTmean to optimally distinguish between GBM and other malignant gliomas was 12.8%, offering 100% sensitivity and 83.3% specificity. These values for APTmean broadly matched the tumor-to-contralateral normal brain tissue ratio from 11C-Met-PET analysis (r = 0.66). The APT signal was also observed in the gadolinium non-contrast region on T1-weighted imaging, appearing to reflect the surrounding tumor-infiltrated area. APT imaging can be used to evaluate the area of tumor invasion, similar to 11C-Met-PET. APT imaging revealed low invasiveness in patients and was useful in preoperative planning for tumor resection, facilitating maximum tumor resection including the tumor invasive area.

Intratumoral thrombosis as a histological biomarker for predicting epidermal growth factor receptor alteration and poor prognosis in patients with glioblastomas.

Intratumoral thrombosis is a specific finding in glioblastomas and considered the origin of palisading necrosis. Its distribution and contribution to the glioblastoma pathophysiology and systemic thrombosis are obscure, although deep vein thrombosis is a common complication in glioblastoma cases. Clinicopathological and genetic analyses were performed on 97 glioblastoma tissue specimens to elucidate the role of thrombotic events and associated molecular abnormalities. Morphologically, intratumoral thrombosis was observed more frequently in vessels composed of single-layered CD34-positive endothelium and/or αSMA-positive pericytes in the tumor periphery, compared to microvascular proliferation with multi-channeled and pericyte-proliferating vessels in the tumor center. Intratumoral thrombosis was significantly correlated with the female sex, high preoperative D-dimer levels, and epidermal growth factor receptor (EGFR) amplification. The presence of one or more thrombi in 20 high-power fields was a predictive marker of EGFR amplification, with a sensitivity of 81.5% and specificity of 52.6%. RNA sequencing demonstrated that the group with many thrombi had higher EGFR gene expression levels than the group with few thrombi. The tumor cells invading along the vessels in the tumor periphery were positive for wild-type EGFR but negative for EGFRvIII, whereas the cells around the microvascular proliferation (MVP) in the tumor center were positive for both wild-type EGFR and EGFRvIII. Intratumoral thrombosis is an independent poor prognostic factor. Aberrant but exquisitely regulated EGFR can induce thrombosis in non-MVP vessels in the tumor invasion area and then promote palisading necrosis, followed by hypoxia, abnormal angiogenesis, and further tumor cell invasion.

MEK inhibitor sensitivity in BRAF fusion-driven prostate cancer.

The identification of subpopulations harboring druggable targets has become a major step forward in the subclassification of solid tumors into small groups suitable for specific therapies. BRAF fusions represent a paradigm of uncommon and targetable oncogenic events and have been widely correlated to the development of specific malignancies. However, they are only present in a limited frequency across most common tumor types. At this regard, we performed a genomic screening aimed to identifying rare variants associated to advanced prostate cancer development. Tumoral tissue genomic screening of 41 patients developing advanced prostate cancer was performed at our center as part of the GETHI XX study. The project, sponsored by the Spanish Collaborative Group in Rare Cancers (GETHI), aims to analyze the molecular background of rare tumors and to discover unfrequent molecular variants in common tumors. Here we present the clinical outcome and an in-deep molecular analysis performed in a case harboring a SND1-BRAF fusion gene. The identification of such rearrangement in a patient refractory to standard therapies led to the administration of trametinib (MEK inhibitor). Despite unsensitive to standard therapies, the patient achieved a dramatic response to trametinib. A comprehensive study of the tumor demonstrated this event to be a trunk alteration with higher expression of MEK in areas of tumor invasion. Our study describes the patient-driven discovery of the first BRAF fusion-driven prostate cancer effectively treated with trametinib. Consequently, MAPK pathway activation could define a new subtype of prostate cancer susceptible to a tailored management.

Prediction of Glioma Stemlike Cell Infiltration in the Non–Contrast-Enhancing Area by Quantitative Measurement of Lactate on Magnetic Resonance Spectroscopy in Glioblastoma

We previously reported that glioma stemlike cells (GSCs) exist in the area of the tumor periphery showing no gadolinium enhancement on magnetic resonance imaging. In the present work, we analyzed glucose metabolism to investigate whether lactate could be predictive of tumor invasiveness and of use in detection of the tumor invasion area in glioblastoma multiforme (GBM). The expression of lactate dehydrogenase A (LDH-A) and pyruvate dehydrogenase (PDH) was investigated in 20 patients. In GSC lines, LDH-A and PDH expression also was examined in parallel to assessments of mitochondrial respiration. We then investigated the relationship between lactate/creatine ratios in the tumor periphery measured by magnetic resonance spectroscopy, using learning-compression-model algorithms and phenotypes of GBMs. In 20 GBMs, high-invasive GBM expressed LDH-A at significantly higher expression than did low-invasive GBM, whereas low-invasive GBM showed significantly higher expression of PDH than did high-invasive GBM. The highly invasive GSC line showed higher expression of LDH-A and lower expression of PDH compared with low-invasive GSC lines. The highly invasive GSC line also showed the lowest consumption of oxygen and the lowest production of adenosine triphosphate. Lactate levels, as measured by magnetic resonance spectroscopy, showed a significant positive correlation with LDH-A transcript levels, permitting classification of the GBMs into high-invasive and low-invasive phenotypes based on a cutoff value of 0.66 in the lactate/creatine ratio. In the tumor periphery area of the highly invasive GBM, aerobic glycolysis was the predominant pathway for glucose metabolism, resulting in the accumulation of lactate. The level of lactate may facilitate prediction of the tumor-infiltrating area on GBM.

Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, microvascular proliferation, mesenchymal transformation and necrosis characterize the histological heterogeneity of high-grade gliomas. Our laboratory has demonstrated that the presence of high densities of mesenchymal cells, named oncostreams, correlate with tumor malignancy. We have developed a unique approach to understand the mechanisms that underlie glioma's growth and invasion. Here, we describe a comprehensive protocol that utilizes laser capture microdissection (LMD) and RNA sequencing to analyze differential mRNA expression of intra-tumoral heterogeneous multicellular structures (i.e., mesenchymal areas or areas of tumor invasion). This method maintains good tissue histology and RNA integrity. Perfusion, freezing, embedding, sectioning, and staining were optimized to preserve morphology and obtain high-quality laser microdissection samples. The results indicate that perfusion of glioma bearing mice using 30% sucrose provides good morphology and RNA quality. In addition, staining tumor sections with 4% Cresyl violet and 0.5% eosin results in good nuclear and cellular staining, while preserving RNA integrity. The method described is sensitive and highly reproducible and it can be utilized to study tumor morphology in various tumor models. In summary, we describe a complete method to perform LMD that preserves morphology and RNA quality for sequencing to study the molecular features of heterogeneous multicellular structures within solid tumors.

P14.129 Predicting glioblastoma invasion using multiparametric MRI and a bi-level machine learning approach

Abstract BACKGROUND Glioblastoma is characterized by its extensive infiltration into the brain parenchyma, which poses challenges to accurate treatment volume targeting. Current treatment planning is based on the contrast-enhancing images. Tumor invasion, however, is well known beyond the contrast enhancement. Although deep learning shows potential in delineating tumor invasion based on multiparametric MRI, it requires large amount of training data. Image inpainting may reconstruct the lost image information based on image structure and requires less training data. The purpose of this study is to investigate whether a bi-level deep learning approach could integrate conventional neural network (CNN) and image inpainting to delineate tumor invasion based on the multiparametric MRI. MATERIAL AND METHODS A total of 115 patients (mean age 59.3 yrs, range 22 - 76 yrs, 87 males) with supratentorial de novo glioblastoma were recruited for maximal safe tumor resection. Post-operative temozolomide chemoradiotherapy was performed following the Stupp protocol. The Response Assessment in Neuro-oncology criteria was used to evaluate patient response. All MRI sequences were pre-operatively acquired using a 3T scanner, including T2-weighted, post-contrast T1-weighted, FLAIR, dynamic susceptibility contrast (DSC) and diffusion tensor imaging (DTI). The isotropic (p) and anisotropic (q) maps were generated from DTI. The relative cerebral blood volume (rCBV), mean transit time (MTT) and relative cerebral blood flow (rCBF) were generated from the DSC images after the leakage correction. All images were coregistered to post-contrast T1W images. Contrast-enhancing (CE) and non-enhancing (NE) regions were semi-automatically segmented and validated by an experienced neuro-radiologist. A tissue segmentation was performed using the FAST (FMRIB’s Automated Segmentation Tool) to segment the normal-appearing brain excluding the above both tumor regions and were classified into three tissue types (grey matter, white matter and CSF). A CNN model was applied to build a classification function between the CE region and the three normal-appearing tissue types based on the multi-parametric MRI. A bespoke inpainting scheme was then applied to predict the tumor invasion area in the NE region. Kaplan-Meier and Cox proportional regression were used to evaluate the significance of the predicted volume. RESULTS The volumes of the contrast-enhancing tumor is 53.6 ± 33.8 cm3. The predicted invasive volume in the non-enhancing region is 31.0 ± 17.7 cm3.The multivariate model shows that the predicted volume was significantly associated with OS (HR = 0.97, P= 0.006). The patient subgroup with a higher predicted volume showed significantly better OS (P= 0.009) and PFS (P= 0.021). CONCLUSION The proposed bi-level deep learning approach may effectively integrate multiparametric MRI for predicting tumor invasio.

The utility of longitudinal slicing method for rectal specimen: pathological analysis of circumferential resection margin of intersphincteric resection for low-lying rectal cancer.

The pathological assessment of the resection margin of rectal cancer is important to predict clinical outcome. The transverse slicing method of rectal specimens is recommended in Western countries. However, in Japan the longitudinal slicing method is traditionally advocated. The aim of this study was to assess the advantages of the longitudinal slicing method. The subjects were 197 consecutive patients with primary rectal cancer who underwent curative intersphincteric resection from 2000 to 2013. The resected rectal specimens were cut into 12 slices in the direction of the long axis. Resection margin was considered positive when it was less than or equal to 1 mm. Resection margin was positive in 23 patients (12%). They were classified into two groups, namely the DEEP group (n = 16, 70%), when the resection margin corresponded to the deepest tumor invasion area, and the ENTRY group (n = 7, 30%), when resection margin was around the initial cutting point of the anal canal. It was shown that resection margin tends to be positive not only in the deepest tumor invasion area but also in the entry area of the anal canal. The longitudinal slicing method may have some advantages for accurate assessment of resection margin especially in low-lying rectal cancer.

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According to the literature regarding the intraoral ultrasonography application for the diagnosis of tongue carcinoma, a tumor invasion area had been demonstrated as hypoechoic compared with the surrounding structure. However, especially with the recently released high-resolution intraoral probe, histopathological invasion front did not always correspond with the deepest margin of the hypoechoic area because the invasion front always tended to be affected by various degree of inflammatory reaction.

The difference in relationship between 18F-FDG uptake and clinicopathological factors on thyroid, esophageal, and lung cancers

The aim of this study was to reveal the differences in clinicopathological factors affecting maximum standardized uptake value (SUVmax) between esophageal squamous cell carcinoma (ESCC), non-small-cell lung cancer (NSCLC), and papillary thyroid cancer (PTC). This study consisted of 119 patients with ESCC (n=43), PTC (n=40), or NSCLC (n=36). We investigated the correlations between SUVmax and clinicopathological factors by using Spearman's correlation coefficient and the Kruskal-Wallis test. Multiple regression analysis was used to investigate which clinicopathological factors significantly affected SUVmax in each cancer type. The SUVmax correlated with glucose transporter-1 (GLUT-1) expression in NSCLC (r=0.536, P=0.007) and ESCC (r=0.597, P<0.001) but not in PTC. The SUVmax correlated with Ki-67 expression in NSCLC (r=0.381, P=0.022) and PTC (r=0.374, P=0.017) but not in ESCC. A high SUVmax was correlated with a higher pathological T stage (p-T stage) in NSCLC (r=0.536) and ESCC (r=0.597, both P<0.001) but not in PTC. An elevated SUVmax was significantly associated with pathological lymph node status (p-N) in NSCLC, but not in ESCC and PTC. In multiple regression analysis, p-T stage and GLUT-1 expression were statistically significant factors in ESCC, and p-T stage was a statistically significant factor in NSCLC. In PTC, Ki-67 showed a statistically significant association with SUVmax. SUVmax in NSCLC depended on the tumor invasion area; SUVmax in ESCC depended on tumor depth and GLUT-1 expression; and SUVmax in PTC might be associated with cell proliferation. The biological factors affecting SUVmax differ according to tumor type.

The Expression of Cyclooxygenase 2 in Retinoblastoma: Primary Enucleated Eyes and Enucleation After Conservative Treatment

To provide insight into the relationship between cyclooxygenase-2 (COX-2) expression and histopathologic features of retinoblastoma specimens treated either by primary or secondary enucleation. Laboratory investigation. Twenty-five retinoblastoma specimens received between 1994 and 2003 were retrieved for this study from the Ocular Pathology Registry, Federal University of São Paulo, Brazil and the Henry C. Witelson Ocular Pathology Laboratory and Registry, McGill University, Montreal, Canada. The specimens retrieved were divided into two groups: Group I, enucleation was performed as a form of primary treatment (n = 15) and Group II, enucleation after failure of conservative treatment (n = 10). Patient information and formalin-fixed, paraffin-embedded specimens were obtained. New sections of these blocks were used for hematoxylin and eosin stain and for immunoassay using a monoclonal mouse anti-COX-2 antibody. Two independent ophthalmic pathologists reviewed all of the microslides. Twenty-three specimens (92%) presented a high expression of COX-2 (15 in Group I; eight in Group II) and there was no statistical difference in COX-2 expression between the two groups (P = .07). However, all specimens expressed COX-2 to a different degree. The areas of tumor invasion were positive for COX-2 in 87.5% of the two groups. Most retinoblastoma specimens revealed a high COX-2 expression. Future studies will be necessary to correlate the high expression of COX-2 in retinoblastoma and a possible applicability of anti-COX-2 medications in the treatment of these tumors.

Expression of Vascular Endothelial Growth Factor Induces an Invasive Phenotype in Human Squamous Cell Carcinomas

Inhibition of the vascular endothelial growth factor (VEGF) receptor Flk-1 has been shown to prevent invasion of experimental squamous cell carcinomas (SCC). To directly investigate the role of VEGF in tumor invasion, we stably transfected human SCC-13 cells, which are characterized by a noninvasive phenotype in vivo, with expression vectors containing murine VEGF(164) in sense (SCC/VEGF+) or antisense (SCC/VEGF-) orientation or with vector alone (SCC/vec). SCC/vec cells formed slowly growing, well-differentiated tumors with well-defined borders between tumor and stroma, after intradermal or subcutaneous injection. In contrast, SCC/VEGF+ tumors were characterized by rapid tumor growth, with small cell groups and single cells invading into the surrounding tissue, and by admixture of blood vessels and tumor cells in areas of tumor invasion. We detected an increase in tumor vessel density and size in VEGF-overexpressing tumors, resulting in a more than fourfold increase in total vascular areas. In contrast, SCC/VEGF- clones formed noninvasive, sharply circumscribed tumors with reduced vascular density. These findings demonstrate that selective VEGF overexpression was sufficient to induce tumor invasiveness, and they provide further evidence for an active role of the tumor stroma in cancer progression.

Relationship of lymphoplasmacytic infiltration of skin tumors to tumor ulceration.

The lymphoplasmacytic infiltrates of 400 benign, borderline, and malignant skin tumors were assessed by light microscopy to determine the relationship between the presence of tumor ulceration and the histologic character of the infiltrate. Squamous cell carcinomas had the highest overall number of infiltrating lymphoplasmacytic cells, while seborrheic keratoses had the lowest. Plasma cells in the infiltrates were related significantly to tumor ulceration (P less than .001). This suggests that the plasma cell infiltrates may be more related to tumor ulceration than to a tumor-specific response in these carcinomas. Small lymphocytes were most evident adjacent to areas of irregularity in the peripheral palisade or of squamous differentiation of the basal cell carcinomas. This suggests that the presence of small lymphocytes, not plasma cells, is a response by the immunocompetent host to areas of tumor invasion.