In Vivo Simvastatin and Brain Radiation in a Model of HER2 + Inflammatory Breast Cancer Brain Metastasis.

Purpose: Inhibiting HMG-CoA reductase with simvastatin prevents breast cancer metastases in preclinical models and radiosensitizes monolayer and stem-like IBC cell lines in vitro . Given the extensive use of simvastatin worldwide and its expected penetration into the brain, we examined whether regulating cholesterol with simvastatin affected IBC3 HER2+ brain metastases. Methods and Materials: Breast cancer cell lines KPL4 and MDA-IBC3 were examined in vitro for DNA repair after radiation with or without statin treatment. Brain metastasis endpoints were examined in the MDA-IBC3 brain metastasis model after ex vivo exposure to lipoproteins and after tail vein injections with and without whole-brain radiotherapy (WBR) and oral statin exposure. Results: Ex vivo preculture of MDA-IBC3 cells with very low-density lipoprotein (vLDL) enhanced the growth of colonized lesions in the brain in vivo compared with control or high-density lipoprotein (HDL), and concurrent oral simvastatin/ WBR reduced the incidence of micrometastatic lesions evaluated 10 days after WBR. However, statin, with or without WBR, did not reduce the incidence, burden, or number of macrometastatic brain lesions evaluated 5 weeks after WBR. Conclusions: Although a role for cholesterol biosynthesis is demonstrated in DNA repair and response to whole brain radiation in this model, durable in vivo efficacy of concurrent whole brain irradiation and oral statin was not demonstrated.

Tumoral Interferon Beta Induces an Immune-Stimulatory Phenotype in Tumor-Associated Macrophages in Melanoma Brain Metastases.

Type I interferons (IFN) are immune-stimulatory cytokines involved in antiviral and antitumor immune responses. They enhance the efficacy of immunogenic anticancer therapies such as radiotherapy by activating both innate and adaptive immune cells. Macrophages are one of the most abundant innate immune cells in the immune microenvironment of melanoma brain metastases (MBM) and can exert potent immune-suppressive functions. Here, we investigate the potential of tumoral type I IFNs to repolarize tumor-associated macrophages (TAM) in two murine MBM models and assess the effects of radiotherapy-induced type I IFN on TAMs in a transcriptomic MBM patient dataset. In mice, we describe a proinflammatory M1-like TAM phenotype induced by tumoral IFNß and identify a myeloid type I IFN-response signature associated with a high M1/M2-like TAM ratio. Following irradiation, patients with MBM displaying a myeloid type I IFN-response signature showed increased overall survival, providing evidence that tumoral IFNß supports an effective antitumor immune response by re-educating immune-regulatory TAM. These findings uncover type I IFN-inducing therapies as a potential macrophage-targeting therapeutic approach and provide a rationale for combining radiotherapy with concomitant immunotherapy to improve treatment response in patients with MBM. SIGNIFICANCE: Our study shows that re-education of tumor-associated macrophages by tumoral IFNß translates into improved clinical outcome in patients with melanoma brain metastases, providing pathomechanistic insights into synergistic type I interferon-inducing therapies with immunotherapies and warranting investigation of IFNß as a predictive biomarker for combined radioimmunotherapy.

Unveiling common transcriptomic features between melanoma brain metastases and neurodegenerative diseases.

Melanoma represents a critical clinical challenge due to its unfavorable outcomes. This type of skin cancer exhibits unique adaptability to the brain microenvironment, but its underlying molecular mechanisms are poorly understood. Recent findings have suggested that melanoma brain metastases (MBM) may share biological processes similar to those found in various neurodegenerative diseases. To further characterize MBM development, we explore the relationship between the transcriptional profiles of MBM and the neurodegenerative diseases Alzheimer's disease, Parkinson's disease, and multiple sclerosis. We take an in silico approach to unveil a neurodegenerative signature of MBM when compared to melanoma non-brain metastasis (53 dysregulated genes enriched in 11 functional terms, such as associated terms to the extracellular matrix and development) and to non tumor-bearing brain controls (195 dysregulated genes, mostly involved in development and cell differentiation, chromatin remodeling and nucleosome organization, and translation). Two genes, ITGA10 and DNAJC6, emerged as key potential markers being dysregulated in both scenarios. Lastly, we developed an open source, user-friendly web tool (https://bioinfo.cipf.es/metafun-mbm/) that allows interactive exploration of the complete results.

Microenvironmental Landscape of Human Melanoma Brain Metastases in Response to Immune Checkpoint Inhibition.

Melanoma-derived brain metastases (MBM) represent an unmet clinical need because central nervous system progression is frequently an end stage of the disease. Immune checkpoint inhibitors (ICI) provide a clinical opportunity against MBM; however, the MBM tumor microenvironment (TME) has not been fully elucidated in the context of ICI. To dissect unique elements of the MBM TME and correlates of MBM response to ICI, we collected 32 fresh MBM and performed single-cell RNA sequencing of the MBM TME and T-cell receptor clonotyping on T cells from MBM and matched blood and extracranial lesions. We observed myeloid phenotypic heterogeneity in the MBM TME, most notably multiple distinct neutrophil states, including an IL8-expressing population that correlated with malignant cell epithelial-to-mesenchymal transition. In addition, we observed significant relationships between intracranial T-cell phenotypes and the distribution of T-cell clonotypes intracranially and peripherally. We found that the phenotype, clonotype, and overall number of MBM-infiltrating T cells were associated with response to ICI, suggesting that ICI-responsive MBMs interact with peripheral blood in a manner similar to extracranial lesions. These data identify unique features of the MBM TME that may represent potential targets to improve clinical outcomes for patients with MBM.

Prophylactic cranial irradiation reduces the incidence of brain metastasis in a mouse model of metastatic, HER2-positive breast cancer.

Prophylactic cranial irradiation (PCI) can reduce the incidence of brain metastasis and improve overall survival in some patients with acute lymphoblastic leukemia or small-cell lung cancer. We examined the potential effects of PCI in a mouse model of breast cancer brain metastasis. The HER2+ inflammatory breast cancer cell line MDA-IBC3 was labeled with green fluorescent protein and injected via tail-vein into female SCID/Beige mice. Mice were then given 0 Gy or 4 Gy of whole-brain irradiation 2 days before tumor-cell injection or 5 days, 3 weeks, or 6 weeks after tumor-cell injection. Mice were sacrificed 4-weeks or 8-weeks after injection and brain tissues were examined for metastasis by fluorescent stereomicroscopy. In the unirradiated control group, brain metastases were present in 77% of mice at 4 weeks and in 90% of mice at 8 weeks; by comparison, rates for the group given PCI at 5 days after tumor-cell injection were 20% at 4 weeks (p=0.01) and 30% at 8 weeks (p=0.02). The PCI group also had fewer brain metastases per mouse at 4 weeks (p=0.03) and 8 weeks (p=0.006) versus the unirradiated control as well as a lower metastatic burden (p=0.01). Irradiation given either before tumor-cell injection or 3-6 weeks afterward had no significant effect on brain metastases compared to the unirradiated control. These results underscore the importance of timing for irradiating subclinical disease. Clinical whole brain strategies to target subclinical brain disease as safely as possible may warrant further study.

Clinical, molecular, metabolic, and immune features associated with oxidative phosphorylation in melanoma brain metastases.

BACKGROUND: Recently, we showed that melanoma brain metastases (MBMs) are characterized by increased utilization of the oxidative phosphorylation (OXPHOS) metabolic pathway compared to melanoma extracranial metastases (ECMs). MBM growth was inhibited by a potent direct OXPHOS inhibitor, but observed toxicities support the need to identify alternative therapeutic strategies. Thus, we explored the features associated with OXPHOS to improve our understanding of the pathogenesis and potential therapeutic vulnerabilities of MBMs. METHODS: We applied an OXPHOS gene signature to our cohort of surgically resected MBMs that had undergone RNA-sequencing (RNA-seq) (n = 88). Clustering by curated gene sets identified MBMs with significant enrichment (High-OXPHOS; n = 21) and depletion (Low-OXPHOS; n = 25) of OXPHOS genes. Clinical data, RNA-seq analysis, and immunohistochemistry were utilized to identify significant clinical, molecular, metabolic, and immune associations with OXPHOS in MBMs. Preclinical models were used to further compare melanomas with High- and Low-OXPHOS and for functional validation. RESULTS: High-OXPHOS MBMs were associated with shorter survival from craniotomy compared to Low-OXPHOS MBMs. High-OXPHOS MBMs exhibited an increase in glutamine metabolism, and treatment with the glutaminase inhibitor CB839 improved survival in mice with MAPKi-resistant, High-OXPHOS intracranial xenografts. High-OXPHOS MBMs also exhibited a transcriptional signature of deficient immune activation, which was reversed in B16-F10 intracranial tumors with metformin treatment, an OXPHOS inhibitor. CONCLUSIONS: OXPHOS is associated with distinct clinical, molecular, metabolic, and immune phenotypes in MBMs. These associations suggest rational therapeutic strategies for further testing to improve outcomes in MBM patients.

Isthmus Incidence in Human Permanent Mandibular First Molars of a South Indian Population: A Cone-Beam Computerized Tomographic Study.

AIM: This study aimed to analyze incidence of isthmus in human permanent mandibular first molar teeth using cone-beam computed tomographic imaging techniques in a South Indian population. MATERIALS AND METHODS: Three hundred permanent mandibular first molar teeth were collected, cleaned, and stored in normal saline. They were divided into groups (GPs) I and II based on number of roots, and were further subdivided (right and left [RL] subgroups A and B for GP I; and RL subgroups C and D for GP-II). Samples were processed and isthmus incidence was evaluated by cone-beam tomography, compared, and statistically analyzed. RESULTS: Overall in mandibular first molars, the isthmus incidence in mesial root was 97.2%, distal root was 39%, and distolingual root was 0%. There was no statistically significant difference between the right and left mandibular first molar teeth with regard to incidence of isthmus (P > 0.05). There was an incidence of type I (38.67%), type II (56.33%), type III (3%), and type IV (2%) isthmuses in mesial root and type I (12.33%), type II (16%), and type III (10.67%) in distal root. CONCLUSION: Incidence of isthmus was very high in the mesial root of the mandibular first molar and should be factored during nonsurgical and surgical endodontic treatment procedures to achieve successful treatment outcomes.

Gene set analysis of post-lactational mammary gland involution gene signatures in inflammatory and triple-negative breast cancer.

BACKGROUND: Epidemiological studies have found that triple-negative breast cancer (TNBC) and TN inflammatory breast cancer (IBC) are associated with lower frequency and duration of breast-feeding compared to non-TNBC and non-TN IBC, respectively. Limited breast-feeding could reflect abrupt or premature involution and contribute to a "primed" stroma that is permissive to the migration of cancer cells typical of IBC. We hypothesized that gene expression related to abrupt mammary gland involution after forced weaning may be enriched in the tissues of IBC patients and, if so, provide a potential correlation between limited breast-feeding and the development of aggressive breast cancer. METHODS: We utilized the Short Time-series Expression Miner (STEM) program to cluster significant signatures from two independent studies that analyzed gene expression at multiple time-points of mouse mammary gland involution. Using 10 significant signatures, we performed gene ontology analysis and gene set enrichment analysis (GSEA) on training and validation sets from human breast cancer gene expression data to identify specific genes that are enriched in IBC compared to non-IBC and in TN compared to non-TN in IBC and non-IBC groups. RESULTS: Examining the combined data, we identified 10 involution gene clusters (Inv1-10) that share time-dependent regulation after forced weaning. Inv5 was the only cluster significantly enriched in IBC in the training and validation set (nominal p-values <0.05) and only by unadjusted p-values (FDR q-values 0.26 and 0.46 respectively). Eight genes in Inv5 are upregulated in both the training and validation sets in IBC. Combining the training and validation sets, both Inv5 and Inv6 have nominal p-values <0.05 and q-values 0.39 and 0.20, respectively. The time course for both clusters includes genes that change within 12 hours after forced weaning. CONCLUSIONS: Results from this in silico study suggest correlation between molecular events during abrupt involution and aggressive breast cancer. Specifically, candidate genes from Inv5 merit functional investigation regarding the role of limited breast-feeding in IBC development.

Assessment of Systemic Inflammatory Markers in Patients with Aggressive Periodontitis.

BACKGROUND: "Aggressive periodontitis (AgP) is a destructive disease characterized by the following: The involvement of multiple teeth with a distinctive pattern of periodontal tissue loss; a high rate of disease progression; an early age of onset; and the absence of systemic diseases.'' Chronic low-level bacteremia and systemic inflammatory response have been suggested as a pathogenic link between periodontal disease and systemic disease. The present study was aimed to assess the levels of systemic inflammatory markers in patients with AgP. METHODS: A sample of 50 systemically healthy patients comprised two groups, based on full mouth periodontal examination: Group I healthy individuals, includes 25 periodontally healthy subjects with fully functioning dentition. Group II includes 25 patients diagnosed clinically as AgP. Laboratory blood investigation included white blood cell (WBC) count, neutrophil count, lymphocyte count, and platelet count. Serum protein parameters included total protein (TP), albumin (ALB), and globulin (GLB). Periodontal clinical parameters including plaque index, gingival index, probing pocket depth, and clinical attachment level were recorded. RESULTS: Data analysis shows an increase in WBC, neutrophil, lymphocyte, and platelet count and a decrease in TP, ALB, and GLB in AgP patients when compared to healthy individuals. CONCLUSION: Results of the present study shows an increase in blood parameters and decrease in serum protein parameters in AgP. Hence, AgP could be considered as one of the risk factors associated with the cardiovascular diseases as assessed by changes in the level of systemic inflammatory markers observed.