Enhanced efficacy of ß-carotene loaded solid lipid nanoparticles optimized and developed via central composite design on breast cancer cell lines.

ß-carotene is obtained from both plants and animals and has been the subject of intense research because of its provitamin-A, antioxidant, and anticancer effects. Its limited absorption and oxidative degradation significantly reduce its antitumor efficacy when taken orally. In our study, we utilize a central composite design to develop "bio-safe and highly bio-compatible" solid lipid nanoparticles (SLNs) by using only the combination of palmitic acid and poloxamer-407, a block co-polymer as a surfactant. The current research aim to develop and characterize SLNs loaded with ß-carotene to improve their bioavailability and therapeutic efficacy. In addition, the improved cytotoxicity of solid lipid nanoparticles loaded with ß-carotene was screened in-vitro in human breast cancer cell lines (MCF-7). The nanoparticles exhibits good stability, as indicated by their mean zeta potential of -26.3 ± 1.3 mV. The particles demonstrated high drug loading and entrapment capabilities. The fabricated nanoparticle's prolonged release potential was shown by the in-vitro release kinetics, which showed a first-order release pattern that adhered to the Higuchi model and showed a slow, linear, and steady release over 48 h. Moreover, a diffusion-type release mechanism was used to liberate ß-carotene from the nanoparticles. For six months, the nanoparticles also showed a notable degree of physical stability. Lastly, using the MTT assay, the anti-cancer properties of ß-carotene-loaded solid lipid nanoparticles were compared with intact ß-carotene on MCF-7 cell lines. The cytotoxicity tests have shown that the encapsulation of ß-carotene in the lipid bilayers of the optimized formulation does not interfere with the anti-cancer activity of the drug. When compared to standard ß-carotene, ß-carotene loaded SLNs showed enhanced anticancer efficacy and it is a plausible therapeutic candidate for enhancing the solubility of water-insoluble and degradation-sensitive biotherapeutics like ß-carotene.

Exploring promising natural compounds for breast cancer treatment: in silico molecular docking targeting WDR5-MYC protein interaction.

Cancer is an aberrant differentiation of normal cells, characterized by uncontrolled growth and the potential to acquire invasive and aggressive properties that ultimately lead to metastasis. In the realm of scientific exploration, a multitude of pathways has been investigated and targeted by researchers, among which one specific pathway is recognized as WDR5-MYC. Continuous investigations and research show that WDR5-MYC is a therapeutic target protein. Hence, the discovery of naturally occurring compounds with anticancer properties has been suggested as a rapid and efficient alternative for the development of anticancerous therapeutics. A virtual screening approach was used to identify the most potent compounds from the NP-lib database at the MTiOpenScreen webserver against WDR5-MYC. This process yielded a total of 304 identified compounds. Subsequently, after screening, four potent compounds, namely Estrone (ZINC000003869899), Ethyl-1,2-benzanthracene (ZINC000003157052), Strychnine (ZINC000000119434) and 7H-DIBENZO [C, G] CARBAZOLE (ZINC000001562130), along with a cocrystallized 5-[4-(trifluoromethyl) phenyl]-1H-tetrazole inhibitor (QBP) as a reference ligand, were considered for stringent molecular docking. Thus, each compound exhibited significant docking energy between -8.2 and -7.7 kcal/mol and molecular contacts with essential residue Asn225, Lys250, Ser267 and Lys272 in the active pocket of WDR5-MYC against the QBP inhibitor (the native ligand QBP serves as a reference in the comparative analysis of docked complexes). The results support the potent compounds for drug-likeness and strong binding affinity with WDR5-MYC protein. Further, the stability of the selected compounds was predicted by molecular dynamics simulation (100 ns) contributed by intermolecular hydrogen bonds and hydrophobic interactions. This demonstrates the potential of the selected compounds to be used against breast cancer treatment.Communicated by Ramaswamy H. Sarma.

CDK4/6 Inhibition Sensitizes Intracranial Tumors to PD-1 Blockade in Preclinical Models of Brain Metastasis.

PURPOSE: Brain metastases are associated with high morbidity and are often resistant to immune checkpoint inhibitors. We evaluated whether CDK4/6 inhibitor (CDKi) abemaciclib can sensitize intracranial tumors to programmed cell death protein 1 (PD-1) inhibition in mouse models of melanoma and breast cancer brain metastasis. EXPERIMENTAL DESIGN: Treatment response was evaluated in vivo using immunocompetent mouse models of brain metastasis bearing concurrent intracranial and extracranial tumors. Treatment effect on intracranial and extracranial tumor-immune microenvironments (TIME) was evaluated using immunofluorescence, multiplex immunoassays, high-parameter flow cytometry, and T-cell receptor profiling. Mice with humanized immune systems were evaluated using flow cytometry to study the effect of CDKi on human T-cell development. RESULTS: We found that combining abemaciclib with PD-1 inhibition reduced tumor burden and improved overall survival in mice. The TIME, which differed on the basis of anatomic location of tumors, was altered with CDKi and PD-1 inhibition in an organ-specific manner. Combination abemaciclib and anti-PD-1 treatment increased recruitment and expansion of CD8+ effector T-cell subsets, depleted CD4+ regulatory T (Treg) cells, and reduced levels of immunosuppressive cytokines in intracranial tumors. In immunodeficient mice engrafted with human immune systems, abemaciclib treatment supported development and maintenance of CD8+ T cells and depleted Treg cells. CONCLUSIONS: Our results highlight the distinct properties of intracranial and extracranial tumors and support clinical investigation of combination CDK4/6 and PD-1 inhibition in patients with brain metastases. See related commentary by Margolin, p. 257.

In Vitro and In Silico Studies on 4-Nitroacetophenone Thiosemicarbazone Potential Cytotoxicity Against A549 Cell Lines.

Lung malignancy is a major worldwide issue that occurs due to the dysregulation of various growth factors. Lung cancer has no apparent signs in the early stages, which makes it harder to catch it in time and leads to a higher fatality rate. So, the goal of this work was to create and analyze a novel chemical molecule called 4-nitro acetophenone thiosemicarbazone (4-NAPTSc) against the lung cancer cell line A549 and human non-tumorigenic lung epithelial cell line BAES-2B. The ligand was synthesized by refluxing the reaction mixture of 4-nitro acetophenone and thiosemicarbazide and was further characterized by UV, FTIR, and 1H and 13C NMR and Differential Scanning Calorimetry (DSC) study. Cytotoxicity assay/MTT (3-(4,5-dimethylthiazol-2-yl))2,5-diphenyltetrazolium bromide) was used to evaluate the cytotoxicity of the compound. Epidermal growth factor receptors (EGFR), polo-like kinase-1 (PLK1), and vascular endothelial growth factor receptors (VEGFR) were chosen as the target proteins for molecular docking to find potential ligand binding sites and inhibit their function. A novel yellow-colored crystalline solid has been synthesized. 4-NAPTSc had an IC50 of 2.93 µg/mL against the A549 lung cancer cells. When the dosage is increased from 5 to 15 µg/mL along with time, the cell viability falls. Docking results showed that the compound binds with the targeted proteins' amino acid residues, and the likeness profile of the compound is also favorable. This study reveals that the compound has the potential for further investigation and can be used in multitargeted cancer therapies.

Nanoparticles in pancreatic cancer therapy: a detailed and elaborated review on patent literature.

INTRODUCTION: Nanotechnology may open up new avenues for overcoming the challenges of pancreatic cancer therapy as a broad arsenal of anticancer medicines fail to realize their full therapeutic potential in pancreatic ductal adenocarcinoma due to the formation of multiple resistance mechanisms inside the tumor. Many studies have reported the successful use of various nano formulations in pancreatic cancer therapy. AREAS COVERED: This review covers all the major nanotechnology-based patent litrature available on renowned patent data bases like Patentscope and Espacenet, through the time period of 2007-2022. This is an entirely patent centric review, and it includes both clinical and non-clinical data available on nanotechnology-based therapeutics and diagnostic tools for pancreatic cancer. EXPERT OPINION: For the sake of understanding, the patents are categorized under various formulation-specific heads like metallic/non-metallic nanoparticles, polymeric nanoparticles, liposomes, carbon nanotubes, protein nanoparticles and liposomes. This distinguishes one specific nanoparticle type from another and makes this review a one-of-a-kind comprehensive patent compilation that has not been reported so far in the history of nanotechnological formulations in pancreatic cancer.

Nanodiamond Mediated Molecular Targeting in Pancreatic Ductal Adenocarcinoma: Disrupting the Tumor-stromal Cross-talk, Next Hope on the Horizon?

Pancreatic ductal adenocarcinoma (PDAC) is one of the foremost causes of cancer-related morbidities worldwide. Novel nanotechnology-backed drug delivery stratagems, including molecular targeting of the chemotherapeutic payload, have been considered. However, no quantum leap in the gross survival rate of patients with PDAC has been realized. One of the predominant causes behind this is tumor desmoplasia, a dense and heterogenous stromal extracellular matrix of the tumor, aptly termed tumor microenvironment (TME). It plays a pivotal role in the tumor pathogenesis of PDAC as it occupies most of the tumor mass, making PDAC one of the most stromal-rich cancers. The complex crosstalk between the tumor and dynamic components of the TME impacts tumor progression and poses a potential barrier to drug delivery. Understanding and deciphering the complex cascade of tumorstromal interactions are the need of the hour so that we can develop neoteric nano-carriers to disrupt the stroma and target the tumor. Nanodiamonds (NDs), due to their unique surface characteristics, have emerged as a promising nano delivery system in various pre-clinical cancer models and have the potential to deliver the chemotherapeutic payload by moving beyond the dynamic tumor-stromal barrier. It can be the next revolution in nanoparticle-mediated pancreatic cancer targeting.

An HLA-G/SPAG9/STAT3 axis promotes brain metastases.

Brain metastases (BM) are the most common brain neoplasm in adults. Current BM therapies still offer limited efficacy and reduced survival outcomes, emphasizing the need for a better understanding of the disease. Herein, we analyzed the transcriptional profile of brain metastasis initiating cells (BMICs) at two distinct stages of the brain metastatic cascade-the "premetastatic" or early stage when they first colonize the brain and the established macrometastatic stage. RNA sequencing was used to obtain the transcriptional profiles of premetastatic and macrometastatic (non-premetastatic) lung, breast, and melanoma BMICs. We identified that lung, breast, and melanoma premetastatic BMICs share a common transcriptomic signature that is distinct from their non-premetastatic counterparts. Importantly, we show that premetastatic BMICs exhibit increased expression of HLA-G, which we further demonstrate functions in an HLA-G/SPAG9/STAT3 axis to promote the establishment of brain metastatic lesions. Our findings suggest that unraveling the molecular landscape of premetastatic BMICs allows for the identification of clinically relevant targets that can possibly inform the development of preventive and/or more efficacious BM therapies.

Characterization of the minimal residual disease state reveals distinct evolutionary trajectories of human glioblastoma.

Recurrence of solid tumors renders patients vulnerable to advanced, treatment-refractory disease state with mutational and oncogenic landscape distinctive from initial diagnosis. Improving outcomes for recurrent cancers requires a better understanding of cell populations that expand from the post-therapy, minimal residual disease (MRD) state. We profile barcoded tumor stem cell populations through therapy at tumor initiation, MRD, and recurrence in our therapy-adapted, patient-derived xenograft models of glioblastoma (GBM). Tumors show distinct patterns of recurrence in which clonal populations exhibit either a pre-existing fitness advantage or an equipotency fitness acquired through therapy. Characterization of the MRD state by single-cell and bulk RNA sequencing reveals a tumor-intrinsic immunomodulatory signature with prognostic significance at the transcriptomic level and in proteomic analysis of cerebrospinal fluid (CSF) collected from patients with GBM. Our results provide insight into the innate and therapy-driven dynamics of human GBM and the prognostic value of interrogating the MRD state in solid cancers.

Activity of Adagrasib (MRTX849) in Brain Metastases: Preclinical Models and Clinical Data from Patients with KRASG12C-Mutant Non-Small Cell Lung Cancer.

PURPOSE: Patients with KRAS-mutant non-small cell lung cancer (NSCLC) with brain metastases (BM) have a poor prognosis. Adagrasib (MRTX849), a potent oral small-molecule KRASG12C inhibitor, irreversibly and selectively binds KRASG12C, locking it in its inactive state. Adagrasib has been optimized for favorable pharmacokinetic properties, including long half-life (∼24 hours), extensive tissue distribution, dose-dependent pharmacokinetics, and central nervous system penetration; however, BM-specific antitumor activity of KRASG12C inhibitors remains to be fully characterized. EXPERIMENTAL DESIGN: A retrospective database query identified patients with KRAS-mutant NSCLC to understand their propensity to develop BM. Preclinical studies assessed physiochemical and pharmacokinetic properties of adagrasib. Mice bearing intracranial KRASG12C-mutant NSCLC xenografts (LU99-Luc/H23-Luc/LU65-Luc) were treated with clinically relevant adagrasib doses, and levels of adagrasib in plasma, cerebrospinal fluid (CSF), and brain were determined along with antitumor activity. Preliminary clinical data were collected from 2 patients with NSCLC with untreated BM who had received adagrasib 600 mg twice daily in the phase Ib cohort of the KRYSTAL-1 trial; CSF was collected, adagrasib concentrations measured, and antitumor activity in BM evaluated. RESULTS: Patients with KRAS-mutant NSCLC demonstrated high propensity to develop BM (≥40%). Adagrasib penetrated into CSF and demonstrated tumor regression and extended survival in multiple preclinical BM models. In 2 patients with NSCLC and untreated BM, CSF concentrations of adagrasib measured above the target cellular IC50. Both patients demonstrated corresponding BM regression, supporting potential clinical activity of adagrasib in the brain. CONCLUSIONS: These data support further development of adagrasib in patients with KRASG12C-mutant NSCLC with untreated BM. See related commentary by Kommalapati and Mansfield, p. 3179.

Recent Advancements in Nanodiamond Mediated Brain Targeted Drug Delivery and Bioimaging of Brain Ailments: A Holistic Review.

BACKGROUND: The brain is a vital and composite organ. By nature, the innate make-up of the brain is such that in anatomical parlance, it is highly protected by the "Blood-Brain Barrier", which is a nexus of capillary endothelial cells, basement membrane, neuroglial membrane and glialpodocytes. The same barrier, which protects and isolates the interstitial fluid of the brain from capillary circulation, also restricts the therapeutic intervention. Many standing pharmaceutical formulations are ineffective in the treatment of inimical brain ailments because of the inability of the API to surpass and subsist inside the Blood Brain Barrier. OBJECTIVE: This is an integrated review that emphasizes on the recent advancements in brain-targeted drug delivery utilizing nanodiamonds (NDs) as a carrier of therapeutic agents. NDs are a novel nanoparticulate drug delivery system, having carbon moieties as their building blocks and their surface tenability is remarkable. These neoteric carbon-based carriers have exceptional, mechanical, electrical, chemical, optical, and biological properties, which can be further rationally modified and augmented. DISCUSSION: NDs could be the next"revolution "in the field of nanoscience for the treatment of neurodegenerative disorders, brain tumors, and other pernicious brain ailments. What sets them apart from other nanocarriers is their versatile properties like diverse size range and surface modification potential, which makes them efficient enough to move across certain biological barriers and offer a plethora of brain targeting and bioimaging abilities. CONCLUSION: The blood-brain barrier (BBB) poses a major hurdle in the way of treating many serious brain ailments. A range of nanoparticle based drug delivering systems have been formulated, including solid lipid nanoparticles, liposomes, dendrimers, nanogels, polymeric NPs, metallic NPs (gold, platinum, andironoxide) and diamondoids (carbonnanotubes). Despite this development, only a few of these formulations have shown the ability to cross the BBB. Nanodiamonds, because of their small size, shape, and surface characteristics, have a potential in moving beyond the diverse and intricate BBB, and offer a plethora of brain targeting capabilities.

Temporal profiling of therapy resistance in human medulloblastoma identifies novel targetable drivers of recurrence.

Medulloblastoma (MB) remains a leading cause of cancer-related mortality among children. The paucity of MB samples collected at relapse has hindered the functional understanding of molecular mechanisms driving therapy failure. New models capable of accurately recapitulating tumor progression in response to conventional therapeutic interventions are urgently needed. In this study, we developed a therapy-adapted PDX MB model that has a distinct advantage of generating human MB recurrence. The comparative gene expression analysis of MB cells collected throughout therapy led to identification of genes specifically up-regulated after therapy, including one previously undescribed in the setting of brain tumors, bactericidal/permeability-increasing fold-containing family B member 4 (BPIFB4). Subsequent functional validation resulted in a markedly diminished in vitro proliferation, self-renewal, and longevity of MB cells, translating into extended survival and reduced tumor burden in vivo. Targeting endothelial nitric oxide synthase, a downstream substrate of BPIFB4, impeded growth of several patient-derived MB lines at low nanomolar concentrations.

Preclinical Solid Tumor Models to Study Novel Therapeutics in Brain Metastases.

Metastases are the most common malignancy of the adult central nervous system and are becoming an increasingly troubling problem in oncology largely due to the lack of successful therapeutic options. The limited selection of treatments is a result of the currently poor understanding of the biological mechanisms of metastatic development, which in turn is difficult to achieve because of limited preclinical models that can accurately represent the clinical progression of metastasis. Described in this article are in vitro and in vivo model systems that are used to enhance the understanding of metastasis and to identify new therapies for the treatment of brain metastasis. © 2021 Wiley Periodicals LLC.

Detection of Leptomeningeal Disease Using Cell-Free DNA From Cerebrospinal Fluid.

Importance: Leptomeningeal disease (LMD) is a devastating complication of cancer that is frequently underdiagnosed owing to the low sensitivity of cerebrospinal fluid (CSF) cytologic assessment, the current benchmark diagnostic method. Improving diagnostic sensitivity may lead to improved treatment decisions. Objective: To assess whether cell-free DNA (cfDNA) analysis of CSF may be used to diagnose LMD more accurately than cytologic analysis. Design, Setting, and Participants: This diagnostic study conducted in a neuro-oncology clinic at 2 large, tertiary medical centers assessed the use of genomic sequencing of CSF samples obtained from 30 patients with suspected or confirmed LMD from 2015 through 2018 to identify tumor-derived cfDNA. From the same CSF samples, cytologic analyses were conducted, and the results of the 2 tests were compared. This study consisted of 2 patient populations: 22 patients with cytologically confirmed LMD without parenchymal tumors abutting their CSF and 8 patients with parenchymal brain metastases with no evidence of LMD. Patients were considered positive for the presence of LMD if previous CSF cytologic analysis was positive for malignant cells. The analysis was conducted from 2015 to 2018. Main Outcomes and Measures: The primary outcome was the diagnostic accuracy of cfDNA analysis, defined as the number of tests that resulted in correct diagnoses out of the total number of tests assayed. Hypotheses were formed before data collection. Results: In total, 30 patients (23 women [77%]; median age, 51 years [range, 28-81 years]), primarily presenting with metastatic solid malignant neoplasms, participated in this study. For 48 follow-up samples from patients previously diagnosed via cytologic analysis as having LMD with no parenchymal tumor abutting CSF, cfDNA findings were accurate in the assessment of LMD in 45 samples (94%; 95% CI, 83%-99%), whereas cytologic analysis was accurate in 36 samples (75%; 95% CI, 60%-86%), a significant difference (P = .02). Of 43 LMD-positive samples, CSF cfDNA analysis was sensitive to LMD in 40 samples (93%; 95% CI, 81%-99%), and cytologic analysis was sensitive to LMD in 31 samples (72%; 95% CI, 56%-85%), a significant difference (P = .02). For 3 patients with parenchymal brain metastases abutting the CSF and no suspicion of LMD, cytologic findings were negative for LMD in all 3 patients, whereas cfDNA findings were positive in all 3 patients. Conclusions and Relevance: This diagnostic study found improved sensitivity and accuracy of cfDNA CSF testing vs cytologic assessment for diagnosing LMD with the exception of parenchymal tumors abutting CSF, suggesting improved ability to diagnosis LMD. Consideration of incorporating CSF cfDNA analysis into clinical care is warranted.

Emerging Immunotherapies in the Treatment of Brain Metastases.

Brain metastases account for considerable morbidity and mortality in patients with cancer. Despite increasing prevalence, limited therapeutic options exist. Recent advances in our understanding of the molecular and cellular underpinnings of the tumor immune microenvironment and the immune evasive mechanisms employed by tumor cells have shed light on how immunotherapies may provide therapeutic benefit to patients. The development and evolution of immunotherapy continue to show promise for the treatment of brain metastases. Positive outcomes have been observed in several studies evaluating the efficacy and safety of these treatments. However, many challenges persist in the application of immunotherapies to brain metastases. This review discusses the potential benefits and challenges in the development and use of checkpoint inhibitors, chimeric antigen receptor T-cell therapy, and oncolytic viruses for the treatment of brain metastases. Future studies are necessary to further evaluate and assess the potential use of each of these therapies in this setting. As we gain more knowledge regarding the role immunotherapies may play in the treatment of brain metastases, it is important to consider how these treatments may guide clinical decision making for clinicians and the impact they may have on patients. IMPLICATIONS FOR PRACTICE: Immunotherapies have produced clinically significant outcomes in early clinical trials evaluating patients with brain metastases or demonstrated promising results in preclinical models. Checkpoint inhibitors have been the most common immunotherapy studied to date in the setting of brain metastases, but novel approaches that can harness the immune system to contain and eliminate cancer cells are currently under investigation and may soon become more common in the clinical setting. An understanding of these evolving therapies may be useful in determining how the future management and treatment of brain metastases among patients with cancer will continue to advance.

The Rational Development of CD133-Targeting Immunotherapies for Glioblastoma.

CD133 marks self-renewing cancer stem cells (CSCs) in a variety of solid tumors, and CD133+ tumor-initiating cells are known markers of chemo- and radio-resistance in multiple aggressive cancers, including glioblastoma (GBM), that may drive intra-tumoral heterogeneity. Here, we report three immunotherapeutic modalities based on a human anti-CD133 antibody fragment that targets a unique epitope present in glycosylated and non-glycosylated CD133 and studied their effects on targeting CD133+ cells in patient-derived models of GBM. We generated an immunoglobulin G (IgG) (RW03-IgG), a dual-antigen T cell engager (DATE), and a CD133-specific chimeric antigen receptor T cell (CAR-T): CART133. All three showed activity against patient-derived CD133+ GBM cells, and CART133 cells demonstrated superior efficacy in patient-derived GBM xenograft models without causing adverse effects on normal CD133+ hematopoietic stem cells in humanized CD34+ mice. Thus, CART133 cells may be a therapeutically tractable strategy to target CD133+ CSCs in human GBM or other treatment-resistant primary cancers.

Bmi1 regulates human glioblastoma stem cells through activation of differential gene networks in CD133+ brain tumor initiating cells.

PURPOSE: Glioblastoma (GBM) is the most aggressive adult brain cancer, with a 15 month median survivorship attributed to the existence of treatment-refractory brain tumor initiating cells (BTICs). In order to better understand the mechanisms regulating the tumorigenic properties of this population, we studied the role of the polycomb group member BMI1 in our patient-derived GBM BTICs and its relationship with CD133, a well-established marker of BTICs. METHODS: Using gain and loss-of-function studies for Bmi1 in neural stem cells (NSCs) and patient-derived GBM BTICs respectively, we assessed in vitro self-renewal and in vivo tumor formation in these two cell populations. We further explored the BMI1 transcriptional regulatory network through RNA sequencing of different GBM BTIC populations that were knocked down for Bmi1. RESULTS: There is a differential role of BMI1 in CD133-positive cells, notably involving cell metabolism. In addition, we identified pivotal targets downstream of BMI1 in CD133+ cells such as integrin alpha 2 (ITGA2), that may contribute to regulating GBM stem cell properties. CONCLUSIONS: Our work sheds light on the association of three genes with CD133-BMI1 circuitry, their importance as downstream effectors of the BMI1 signalling pathway, and their potential as future targets for tackling GBM treatment-resistant cell populations.

BMI1 is a therapeutic target in recurrent medulloblastoma.

Medulloblastoma (MB) is the most frequent malignant pediatric brain tumor, representing 20% of newly diagnosed childhood central nervous system malignancies. Although advances in multimodal therapy yielded a 5-year survivorship of 80%, MB still accounts for the leading cause of childhood cancer mortality. In this work, we describe the epigenetic regulator BMI1 as a novel therapeutic target for the treatment of recurrent human Group 3 MB, a childhood brain tumor for which there is virtually no treatment option beyond palliation. Current clinical trials for recurrent MB patients based on genomic profiles of primary, treatment-naive tumors will provide limited clinical benefit since recurrent metastatic MBs are highly genetically divergent from their primary tumor. Using a small molecule inhibitor against BMI1, PTC-028, we were able to demonstrate complete ablation of self-renewal of MB stem cells in vitro. When administered to mice xenografted with patient tumors, we observed significant reduction in tumor burden in both local and metastatic compartments and subsequent increased survival, without neurotoxicity. Strikingly, serial in vivo re-transplantation assays demonstrated a marked reduction in tumor initiation ability of recurrent MB cells upon re-transplantation of PTC-028-treated cells into secondary recipient mouse brains. As Group 3 MB is often metastatic and uniformly fatal at recurrence, with no current or planned trials of targeted therapy, an efficacious targeted agent would be rapidly transitioned to clinical trials.

In Vivo Murine Models of Brain Metastasis.

Metastases are the most common tumor type to affect the adult central nervous system. In vivo modeling of brain metastases provides insight into the mechanisms of metastatic development as well as a clinically relevant therapeutic screening platform. Here we describe the development of a novel mouse model of brain metastasis from a primary lung cancer utilizing primary patient samples. These models provide an accurate representation of different stages of the clinical progression of the disease.

Therapeutic Targeting of the Premetastatic Stage in Human Lung-to-Brain Metastasis.

Brain metastases (BM) result from the spread of primary tumors to the brain and are a leading cause of cancer mortality in adults. Secondary tissue colonization remains the main bottleneck in metastatic development, yet this "premetastatic" stage of the metastatic cascade, when primary tumor cells cross the blood-brain barrier and seed the brain before initiating a secondary tumor, remains poorly characterized. Current studies rely on specimens from fully developed macrometastases to identify therapeutic options in cancer treatment, overlooking the potentially more treatable "premetastatic" phase when colonizing cancer cells could be targeted before they initiate the secondary brain tumor. Here we use our established brain metastasis initiating cell (BMIC) models and gene expression analyses to characterize premetastasis in human lung-to-BM. Premetastatic BMIC engaged invasive and epithelial developmental mechanisms while simultaneously impeding proliferation and apoptosis. We identified the dopamine agonist apomorphine to be a potential premetastasis-targeting drug. In vivo treatment with apomorphine prevented BM formation, potentially by targeting premetastasis-associated genes KIF16B, SEPW1, and TESK2 Low expression of these genes was associated with poor survival of patients with lung adenocarcinoma. These results illuminate the cellular and molecular dynamics of premetastasis, which is subclinical and currently impossible to identify or interrogate in human patients with BM. These data present several novel therapeutic targets and associated pathways to prevent BM initiation.Significance: These findings unveil molecular features of the premetastatic stage of lung-to-brain metastases and offer a potential therapeutic strategy to prevent brain metastases. Cancer Res; 78(17); 5124-34. ©2018 AACR.

Cotargeting Ephrin Receptor Tyrosine Kinases A2 and A3 in Cancer Stem Cells Reduces Growth of Recurrent Glioblastoma.

Glioblastoma (GBM) carries a dismal prognosis and inevitably relapses despite aggressive therapy. Many members of the Eph receptor tyrosine kinase (EphR) family are expressed by GBM stem cells (GSC), which have been implicated in resistance to GBM therapy. In this study, we identify several EphRs that mark a therapeutically targetable GSC population in treatment-refractory, recurrent GBM (rGBM). Using a highly specific EphR antibody panel and CyTOF (cytometry by time-of-flight), we characterized the expression of all 14 EphR in primary and recurrent patient-derived GSCs to identify putative rGBM-specific EphR. EPHA2 and EPHA3 coexpression marked a highly tumorigenic cell population in rGBM that was enriched in GSC marker expression. Knockdown of EPHA2 and EPHA3 together led to increased expression of differentiation marker GFAP and blocked clonogenic and tumorigenic potential, promoting significantly higher survival in vivo Treatment of rGBM with a bispecific antibody against EPHA2/A3 reduced clonogenicity in vitro and tumorigenic potential of xenografted recurrent GBM in vivo via downregulation of AKT and ERK and increased cellular differentiation. In conclusion, we show that EPHA2 and EPHA3 together mark a GSC population in rGBM and that strategic cotargeting of EPHA2 and EPHA3 presents a novel and rational therapeutic approach for rGBM.Significance: Treatment of rGBM with a novel bispecific antibody against EPHA2 and EPHA3 reduces tumor burden, paving the way for the development of therapeutic approaches against biologically relevant targets in rGBM. Cancer Res; 78(17); 5023-37. ©2018 AACR.