In this Perspective, we provide our insights and opinions about the contribution—and potential co-regulation—of mechanics and metabolism in incurable breast cancer brain metastasis. Altered metabolic ...activity can affect cancer metastasis as high glucose supply and demand in the brain microenvironment favors aerobic glycolysis. Similarly, the altered mechanical properties of disseminating cancer cells facilitate migration to and metastatic seeding of the brain, where local metabolites support their progression. Cancer cells in the brain and the brain tumor microenvironment often possess opposing mechanical and metabolic properties compared to extracranial cancer cells and their microenvironment, which inhibit the ease of extravasation and metastasis of these cells outside the central nervous system. We posit that the brain provides a metabolic microenvironment that mechanically reinforces the cellular structure of cancer cells and supports their metastatic growth while restricting their spread from the brain to external organs.
Solid stress and tissue stiffness affect tumour growth, invasion, metastasis and treatment. Unlike stiffness, which can be precisely mapped in tumours, the measurement of solid stresses is ...challenging. Here, we show that 2D spatial maps of the solid stress and the resulting elastic energy in excised or in situ tumours with arbitrary shapes and a wide range of sizes can be obtained via three distinct and quantitative techniques that rely on the measurement of tissue displacement after disruption of the confining structures. Application of these methods in models of primary tumours and metastasis revealed that (i) solid stress depends on both cancer cells and their microenvironments, (ii) solid stress increases with tumour size and (iii) mechanical confinement by the surrounding tissue substantially contributes to intratumoral solid stress. Further study of the genesis and consequences of solid stress, facilitated by the engineering principles presented here, may lead to new discoveries and therapies.Three methods for releasing solid stress in tumours provide two–dimensional mappings, sensitive estimates and in situ quantification of stress-induced tumour deformations — and thus stored elastic energy — via ultrasonography or optical microscopy.
Physiological abnormalities in pulmonary granulomas-pathological hallmarks of tuberculosis (TB)-compromise the transport of oxygen, nutrients, and drugs. In prior studies, we demonstrated ...mathematically and experimentally that hypoxia and necrosis emerge in the granuloma microenvironment (GME) as a direct result of limited oxygen availability. Building on our initial model of avascular oxygen diffusion, here we explore additional aspects of oxygen transport, including the roles of granuloma vasculature, transcapillary transport, plasma dilution, and interstitial convection, followed by cellular metabolism. Approximate analytical solutions are provided for oxygen and glucose concentration, interstitial fluid velocity, interstitial fluid pressure, and the thickness of the convective zone. These predictions are in agreement with prior experimental results from rabbit TB granulomas and from rat carcinoma models, which share similar transport limitations. Additional drug delivery predictions for anti-TB-agents (rifampicin and clofazimine) strikingly match recent spatially-resolved experimental results from a mouse model of TB. Finally, an approach to improve molecular transport in granulomas by modulating interstitial hydraulic conductivity is tested in silico.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Excessive deposition of extracellular matrix (ECM) is a hallmark of solid tumors; however, it remains poorly understood which cellular and molecular components contribute to the formation of ECM ...stroma in central nervous system (CNS) tumors. Here, we undertake a pan-CNS analysis of retrospective gene expression datasets to characterize inter- and intra-tumoral heterogeneity of ECM remodeling signatures in both adult and pediatric CNS disease. We find that CNS lesions - glioblastoma in particular - can be divided into two ECM-based subtypes (ECM
and ECM
) that are influenced by the presence of perivascular stromal cells resembling cancer-associated fibroblasts (CAFs). Ligand-receptor network analysis predicts that perivascular fibroblasts activate signaling pathways responsible for recruitment of tumor-associated macrophages and promotion of cancer stemness. Our analysis reveals that perivascular fibroblasts are correlated with unfavorable response to immune checkpoint blockade in glioblastoma and poor patient survival across a subset of CNS tumors. We provide insights into new stroma-driven mechanisms underlying immune evasion and immunotherapy resistance in CNS tumors like glioblastoma, and discuss how targeting these perivascular fibroblasts may prove an effective approach to improving treatment response and patient survival in a variety of CNS tumors.
BackgroundChimeric antigen receptor (CAR)-T cells has revolutionized the treatment of hematological malignancies.1 However, they have shown limited or no efficacy in patients with glioblastoma (GBM) ...or other solid tumors due to poor infiltration into tumors and immunosuppressive tumor microenvironment (TME).2 3 We previously showed that blocking vascular endothelial growth factor (VEGF) signaling normalizes tumor vessels, reprograms the immunosuppressive TME into an immunostimulatory milieu, and improves the efficacy of immunotherapy.4 5 Here, we tested the hypothesis that anti-VEGF therapy (B20) can improve the delivery and efficacy of CAR-T cells in immunocompetent orthotopic GBM mouse models.MethodsTwo syngeneic mouse GBM cell lines (CT2A and GSC005) were used in the study. They were engineered to express EGFRvIII, one of the most common neoantigens in human GBM.6 7 CAR-T cells were designed to recognize EGFRvIII. Orthotopically injected, GBM-bearing C57BL/6 mice were treated with B20 (2.5 mg/kg, every 3 days for 4 doses), followed by EGFRvIII-CAR-T injection. We used intravital imaging with two-photo microscopy to track the infiltration of CAR-T cells into the tumor and flow cytometry to measure the number and function of CAR-T and other immune cells.ResultsCombination of B20 with CAR-T cell treatment prolonged survival of GBM-bearing mice compared to CAR-T cell therapy alone. Intravital imaging revealed that B20 normalized tumor vasculature (figure 1A) and a combination of B20 increased the number of infiltrated CAR-T cells up to 4-fold compared to the CAR-T therapy without B20 (figure 1B). Flow cytometry analysis resulted in an increased population of IL-2+ or IFN-γ+ CAR-T cells, indicating that B20 increased the anti-tumor function of injected CAR-T cells (figure 1C). Moreover, the population of endogenous lymphocytes was increased after B20 therapy. Increased Granzyme B+ TNF-α+ CD8 T cells (Cytotoxic T lymphocytes; CTLs) and decreased FoxP3+ CD4 T cells (Regulatory T cells; Tregs) were observed after B20 treatment indicating that the TME was remodeled to increase the effect of CAR-T therapy (figure 1D).ConclusionsOur strategy improved the efficacy of CAR-T therapy in GBM mouse models by increasing the CAR-T cell infiltration and reprograming TME by increasing the activation of CAR-T cells and endogenous effector T cells.8 Our findings provide compelling data and a rationale for the clinical evaluation of combining anti-VEGF agents with CAR-T cells for GBM patients. Furthermore, we are expanding this approach to improve CAR-T therapy for breast cancer brain metastasis, which shares similar immunosuppressive brain TME features.AcknowledgementsWe would like to thank Dr. Darrell Irvine of MIT for providing the murine EGFRvIII-CAR and EGFRvIII constructs, Dr. Thomas N. Seyfried of Boston University for providing CT2A cells, and Dr. Samuel D. Rabkin of Massachusetts General Hospital for providing GSC005 cells. We also thank Drs. Heena Kumra, Hye-Jung Kim, Igor dos Santos and Sampurna Chatterjee for their helpful input on this manuscript; and Anna Khachatryan and Carolyn Smith for their technical assistance.ReferencesJune CH, O’Connor RS, Kawalekar OU, Ghassemi S, Milone MC. CAR T cell immunotherapy for human cancer. Science. 2018;359(6382):1361–1365.O’Rourke DM, Nasrallah MP, Desai A, Melenhorst JJ, Mansfield K, Morrissette JJ, ..., Maus MV. A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma. Sci Transl Med. 2017;9(399):eaaa0984.Young RM, Engel NW, Uslu U, Wellhausen N, June CH. Next-generation CAR T-cell therapies. Cancer Discov. 2022;12(7):1625–1633.Amoozgar Z, Ren J, Wang N, Andersson P, Ferraro G, Rajan S, ..., Jain RK. Combined blockade of VEGF, Angiopoietin-2, and PD1 reprograms glioblastoma endothelial cells into quasi-antigen-presenting cells. bioRxiv. 2022; 2022–09: 506476.Huang Y, Yuan J, Righi E, Kamoun WS, Ancukiewicz M, Nezivar J, ..., Poznansky MC. Vascular normalizing doses of antiangiogenic treatment reprogram the immunosuppressive tumor microenvironment and enhance immunotherapy. Proc Natl Acad Sci U S A. 2012;109(43):17561–6.Johnson LA, Scholler J, Ohkuri T, Kosaka A, Patel PR, McGettigan SE, ..., Maus MV. Rational development and characterization of humanized anti-EGFR variant III chimeric antigen receptor T cells for glioblastoma. Sci Transl Med. 2015;7(275):275ra22.Brown CE, Alizadeh D, Starr R, Weng L, Wagner JR, Naranjo A, ..., Badie B. Regression of glioblastoma after chimeric antigen receptor T-cell therapy. N Engl J Med. 2016;375(26):2561–9.Dong X, Ren J, Amoozgar Z, Lee S, Datta M, Roberge S, ..., Jain RK. Anti-VEGF therapy improves EGFR-vIII-CAR-T cell delivery and efficacy in syngeneic glioblastoma models in mice. J Immunother Cancer. 2023;11(3):e005583.Abstract 249 Figure 1(A) Intravital image by multiphoton microscopy of brain tumor vasculature two days after B20 (2.5 mg/kg) treatment. (B) Intratumoral distribution of EGFRvIII-CAR-T cells inside of GFP-GSC005 GBM tumors imaged by multiphoton microscopy. Images were taken 24 hours after the injection of CAR-T cells. Measurement of CAR-T cell (pink) number in GFP-GSC005 tumors (green) treated with PBS or B20 (2.5 mg/kg). (C) Percentage of IL2+and IFN-y+ CAR T cells inside the GSC005 GBM tumors. (D) Percentage of the cytotoxic Granzyme B+TNF-α+ CD8 T cells and regulatory FoxP3+ CD4 T cells in the GSC005 tumors. Error bars show ±SEM. Statistical analysis was performed using Student’s t-test.*p<0.05, **p<0.01 *** , p<0.001.
The GL261 and CT2A syngeneic tumor lines are frequently used as immunocompetent orthotopic mouse models of human glioblastoma (huGBM) but demonstrate distinct differences in their responses to ...immunotherapy.
To decipher the cell-intrinsic mechanisms that drive immunotherapy resistance in CT2A-luc and to define the aspects of human cancer biology that these lines can best model, we systematically compared their characteristics using whole exome and transcriptome sequencing, and protein analysis through immunohistochemistry, Western blot, flow cytometry, immunopeptidomics, and phosphopeptidomics.
The transcriptional profiles of GL261-luc2 and CT2A-luc tumors resembled those of some huGBMs, despite neither line sharing the essential genetic or histologic features of huGBM. Both models exhibited striking hypermutation, with clonal hotspot mutations in RAS genes (
p.G12C in GL261-luc2 and
p.Q61L in CT2A-luc). CT2A-luc distinctly displayed mesenchymal differentiation, upregulated angiogenesis, and multiple defects in antigen presentation machinery (e.g.
p.Y488C and
p.A275P mutations) and interferon response pathways (e.g. copy number losses of loci including IFN genes and reduced phosphorylation of JAK/STAT pathway members). The defect in MHC class I expression could be overcome in CT2A-luc by interferon-γ treatment, which may underlie the modest efficacy of some immunotherapy combinations. Additionally, CT2A-luc demonstrated substantial baseline secretion of the CCL-2, CCL-5, and CCL-22 chemokines, which play important roles as myeloid chemoattractants.
Although the clinical contexts that can be modeled by GL261 and CT2A for huGBM are limited, CT2A may be an informative model of immunotherapy resistance due to its deficits in antigen presentation machinery and interferon response pathways.
BackgroundGlioblastoma (GBM) is a fatal malignancy with a median overall survival of <2 years with the current standard of care.1 Wnt signaling fuels GBM progression by aiding proliferation, stemness ...and chemo-resistance.2–4 Importantly, Wnt is enriched in glioma stem cells (GSCs), that confer resistance to chemotherapy and anti-tumor immunity.5 6 This may partly explain why immune checkpoint blockers (ICBs) have failed in Phase III clinical trials in GBM7 NCT02617589. Thus, Wnt signaling is an appealing therapeutic target to overcome resistance of ICBs in GBM. Here, we tested the hypothesis that Wnt inhibition potentiates anti-PD1 antibody (aPD1) treatment by reprograming the GBM tumor microenvironment (TME) from immuno-suppressive to immune-stimulatory in an orthotopic mouse model rich in GSCs.Methods005GSC-GFP cells were orthotopically implanted in immunocompetent C7BL/6 mice bearing cranial windows. Tumor growth was tracked with ultrasound-based imaging and later randomized into treatment groups of control, WNT974 (a porcupine inhibitor of Wnt signaling; 5 mg/kg, oral gavage, daily), aPD1 (250 ug i.p., once every three days), and WNT974+aPD1. GBM samples were later collected for fluorescence activated cell sorting (FACS) to compare immune cell subpopulations among different treatment groups. Western blot, qPCR, and immunohistochemistry (IHC) analysis was conducted on mouse and human GBM samples to analyze the expression level of Wnt signaling molecules.ResultsThe combination of WNT974 with aPD1 prolonged the median survival of 005GSC-GFP bearing mice compared to the non-treated control group, from 25 days to 59 days (figure 1A). FACS analysis revealed that the WNT974+aPD1 treated group had an increased frequency of DC3-like dendritic cells expressing CCR7, CD80, and CD40+, decreased frequency of CD45+CD11b+Ly6G+Ly6C- granulocytic myeloid-derived suppressor cells (gMDSCs), and increased frequency of CD45+CD11b+Ly6G-Ly6C+ monocytic MDSCs (mMDSCs) compared to the untreated or aPD1 monotherapy mice. The combination therapy did not affect the CD4+ and CD8+ T cells in TME (figure 1C). Molecular and IHC analysis revealed a decreased expression level of Wnt signaling molecules in the WNT974+aPD1 group as compared to that in the non-treated group (figure 1B).ConclusionsWnt inhibition improved the efficacy of ICB therapy in a stem cell rich GBM mouse model by reprogramming the myeloid cell subpopulation in the TME. This combination showed a heterogeneous response making some animals poor responders. We are currently investigating the resistance mechanisms to this combination therapy, and building our previous findings on the role of Wnt signaling on the interaction between the GBM vasculature with GSCs.8 AcknowledgementsWe thank Carolyn Smith for outstanding technical support of the immunohistochemistry studies. We thank Sampurna Chatterjee and William Ho for guidance with the flow acquisition of the DC and MDSC data. We thank Nilesh Talele for the quantification of Iba1+ staining. This work was supported by the National Foundation for Cancer Research; the Ludwig Center at Harvard; the Jane’s Trust Foundation; the Advanced Medical Research Foundation; and the NIH grants P01-CA080124, R35-CA197743 and U01-CA224348 (to Rakesh K. Jain) and R01-CA208205 (to Dai Fukumura and Rakesh K. Jain). Zohreh Amoozgar was supported by Aid for Cancer Research Award, Tosteson Fellowship, and Cancer Center Excellence Award from Massachusetts General Hospital. Shanmugarajan Krishnan was supported by DoD fellowship W81XWH-19-1-0723. JP was supported by NIH Training Grants (grant no. T32HL007627 and T32CA251062). MD was supported by NIH/NCI K22CA258410.ReferencesTan AC, Ashley DM, López GY, Malinzak M, Friedman HS, Khasraw M. Management of glioblastoma: State of the art and future directions. CA: a cancer journal for clinicians. 2020;70:299–312.Kaur N, Chettiar S, Rathod S, Rath P, Muzumdar D, Shaikh ML, Shiras A. Wnt3a mediated activation of Wnt/β-catenin signaling promotes tumor progression in glioblastoma. Molecular and Cellular Neuroscience. 2013;54:44–57.Rheinbay E, Suvà ML, Gillespie SM, Wakimoto H, Patel AP, Shahid M, ..., Bernstein BE. An aberrant transcription factor network essential for Wnt signaling and stem cell maintenance in glioblastoma. Cell Reports. 2013;3:1567–1579.Zhan T, Rindtorff N, Boutros M. Wnt signaling in cancer. Oncogene. 2017;36:1461–1473.Chen P, Hsu WH, Han J, Xia Y, DePinho RA. Cancer stemness meets immunity: from mechanism to therapy. Cell Reports. 2021;34 Luke JJ, Bao R, Sweis RF, Spranger S, Gajewski TF. WNT/β-catenin pathway activation correlates with immune exclusion across human cancers. Clinical Cancer Research. 2019;25:3074–3083.Filley AC, Henriquez M, Dey M. Recurrent glioma clinical trial, CheckMate-143: the game is not over yet. Oncotarget. 2017;8:91779.Griveau A, Seano G, Shelton SJ, Kupp R, Jahangiri A, Obernier K, Krishnan S, ..., Jain RK, Rowitch DH. A Glial Signature and Wnt7 Signaling Regulate Glioma-Vascular Interactions and Tumor Microenvironment. Cancer Cell. 2018;33:874–889.Abstract 897-F Figure 1(A) 005GSC-GFP cells were orthotopically implanted and randomized into control, WNT974, αPD1 and WNT974+αPD1 and survival was monitored. The median survival for each arm. *p<0.05 using Log Rank test for survival study. (B) IHC for Wnt7b Protein (dark) and immunofluorescence for beta-catenin (red) detection were performed on the tumor tissue collected from time-matched manner post treatment with Control, anti-PD1, and the combination (n=5–7 per group). (C) FACS analysis on the tumor tissue collected from different treatment groups to analyze the subpopulation of DC3-like dendritic cell (% from CD45+CD11C+ cells) and myeloid-derived suppressor cells (% from CD45+CD11b+ cells). Error bars show ±SEM. * p<0.05, ** p<0.01. One-Way ANOVA followed by test for multiple comparison of means
Emerging immunotherapeutic approaches have revolutionized the treatment of multiple malignancies. Immune checkpoint blockers (ICBs) have enabled never-before-seen success rates in durable tumor ...control and enhanced survival benefit in patients with advanced cancers. However, this effect is not universal, resulting in responder and nonresponder populations not only between, but also within solid tumor types. Although ICBs are thought to be most effective against tumors with more genetic mutations and higher antigen loads, this is not always the case for all cancers or for all patients within a cancer subtype. Furthermore, debilitating and sometimes deadly immune-related adverse events (irAEs) have resulted from aberrant activation of T-cell responses following immunotherapy. Thus, we must identify new ways to overcome resistance to ICB-based immunotherapies and limit irAEs. In fact, preclinical and clinical data have identified abnormalities in the tumor microenvironment (TME) that can thwart the efficacy of immunotherapies such as ICBs. Here, we will discuss how reprogramming various facets of the TME (blood vessels, myeloid cells, and regulatory T cells Tregs) may overcome TME-instigated resistance mechanisms to immunotherapy. We will discuss clinical applications of this strategic approach, including the recent successful phase III trial combining bevacizumab with atezolizumab and chemotherapy for metastatic nonsquamous non-small cell lung cancer that led to rapid approval by the U.S. Food and Drug Administration of this regimen for first-line treatment. Given the accelerated testing and approval of ICBs combined with various targeted therapies in larger numbers of patients with cancer, we will discuss how these concepts and approaches can be incorporated into clinical practice to improve immunotherapy outcomes.
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