BackgroundChimeric Antigen Receptor (CAR) therapy has had a transformative impact on the treatment of hematologic malignancies1–6 but success in solid tumors remains elusive. We hypothesized solid ...tumors have cell-intrinsic resistance mechanisms to CAR T-cell cytotoxicity.MethodsTo systematically identify resistance pathways, we conducted a genome-wide CRISPR knockout screen in glioblastoma cells, a disease where CAR T-cells have had limited efficacy.7 8 We utilized the glioblastoma cell line U87 and targeted endogenously expressed EGFR with CAR T-cells generated from 6 normal donors for the screen. We validated findings in vitro and in vivo across a variety of human tumors and CAR T-cell antigens.ResultsLoss of genes in the interferon gamma receptor (IFNγR) signaling pathway (IFNγR1, JAK1, JAK2) rendered U87 cells resistant to CAR T-cell killing in vitro. IFNγR1 knockout tumors also showed resistance to CAR T cell treatment in vivo in a second glioblastoma line U251 in an orthotopic model. This phenomenon was irrespective of CAR target as we also observed resistance with IL13Ralpha2 CAR T-cells. In addition, resistance to CAR T-cell cytotoxicity through loss of IFNγR1 applied more broadly to solid tumors as pancreatic cell lines targeted with either Mesothelin or EGFR CAR T-cells also showed resistance. However, loss of IFNγR signaling did not impact sensitivity of liquid tumor lines (leukemia, lymphoma or multiple myeloma) to CAR T-cells in vitro or in an orthotopic model of leukemia treated with CD19 CAR. We isolated the effects of decreased cytotoxicity of IFNγR1 knockout glioblastoma tumors to be cancer-cell intrinsic because CAR T-cells had no observable differences in proliferation, activation (CD69 and LFA-1), or degranulation (CD107a) when exposed to wildtype versus knockout tumors. Using transcriptional profiling, we determined that glioblastoma cells lacking IFNγR1 had lower upregulation of cell adhesion pathways compared to wildtype glioblastoma cells after exposure to CAR T-cells. We found that loss of IFNγR1 reduced CAR T-cell binding avidity to glioblastoma.ConclusionsThe critical role of IFNγR signaling for susceptibility of solid tumors to CAR T-cells is surprising given that CAR T-cells do not require traditional antigen-presentation pathways. Instead, in glioblastoma tumors, IFNγR signaling was required for sufficient adhesion of CAR T-cells to mediate productive cytotoxicity. Our work demonstrates that liquid and solid tumors differ in their interactions with CAR T-cells and suggests that enhancing T-cell/tumor interactions may yield improved responses in solid tumors.AcknowledgementsRCL was supported by T32 GM007306, T32 AI007529, and the Richard N. Cross Fund. ML was supported by T32 2T32CA071345-21A1. SRB was supported by T32CA009216-38. NJH was supported by the Landry Cancer Biology Fellowship. JJ is supported by a NIH F31 fellowship (1F31-MH117886). GG was partially funded by the Paul C. Zamecnik Chair in Oncology at the Massachusetts General Hospital Cancer Center and NIH R01CA 252940. MVM and this work is supported by the Damon Runyon Cancer Research Foundation, Stand Up to Cancer, NIH R01CA 252940, R01CA238268, and R01CA249062.ReferencesMaude SL, et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Engl J Med 2018;378:439–448.Neelapu SS, et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N Engl J Med 2017;377:2531–2544.Locke FL, et al. Long-term safety and activity of axicabtagene ciloleucel in refractory large B-cell lymphoma (ZUMA-1): a single-arm, multicentre, phase 1–2 trial. The Lancet Oncology 2019;20:31–42.Schuster SJ, et al. Chimeric antigen receptor T cells in refractory B-cell lymphomas. N Engl J Med 2017;377:2545–2554.Wang M, et al. KTE-X19 CAR T-cell therapy in relapsed or refractory mantle-cell lymphoma. N Engl J Med 2020;382:1331–1342.Cohen AD, et al. B cell maturation antigen-specific CAR T cells are clinically active in multiple myeloma. J Clin Invest 2019;129:2210–2221.Bagley SJ, et al. CAR T-cell therapy for glioblastoma: recent clinical advances and future challenges. Neuro-oncology 2018;20:1429–1438.Choi BD, et al. Engineering chimeric antigen receptor T cells to treat glioblastoma. J Target Ther Cancer 2017;6:22–25.Ethics ApprovalAll human samples were obtained with informed consent and following institutional guidelines under protocols approved by the Institutional Review Boards (IRBs) at the Massachusetts General Hospital (2016P001219). Animal work was performed according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) (2015N000218 and 2020N000114).
Chimeric antigen receptor (CAR) therapy has had a transformative effect on the treatment of haematologic malignancies
, but it has shown limited efficacy against solid tumours. Solid tumours may have ...cell-intrinsic resistance mechanisms to CAR T cell cytotoxicity. Here, to systematically identify potential resistance pathways in an unbiased manner, we conducted a genome-wide CRISPR knockout screen in glioblastoma, a disease in which CAR T cells have had limited efficacy
. We found that the loss of genes in the interferon-γ receptor (IFNγR) signalling pathway (IFNGR1, JAK1 or JAK2) rendered glioblastoma and other solid tumours more resistant to killing by CAR T cells both in vitro and in vivo. However, loss of this pathway did not render leukaemia or lymphoma cell lines insensitive to CAR T cells. Using transcriptional profiling, we determined that glioblastoma cells lacking IFNγR1 had lower upregulation of cell-adhesion pathways after exposure to CAR T cells. We found that loss of IFNγR1 in glioblastoma cells reduced overall CAR T cell binding duration and avidity. The critical role of IFNγR signalling in susceptibility of solid tumours to CAR T cells is surprising, given that CAR T cells do not require traditional antigen-presentation pathways. Instead, in glioblastoma tumours, IFNγR signalling was required for sufficient adhesion of CAR T cells to mediate productive cytotoxicity. Our work demonstrates that liquid and solid tumours differ in their interactions with CAR T cells and suggests that enhancing binding interactions between T cells and tumour cells may yield improved responses in solid tumours.
Abstract
Though CAR T cell therapy has had success treating hematologic malignancies, these treatments have shown only modest efficacy in solid tumors. Little is known about the necessary molecular ...components required for CAR T cell cytotoxicity, especially in the context of solid tumors. We investigated the role of IFNgR signaling on solid tumors and discovered across tumor types (including glioblastoma, pancreatic, ovarian and lung) loss of IFNgR1 signaling resulted lower CAR T cell cytotoxicity in vitro and that this was irrespective of target antigen (including endogenous EGFR, IL13Ra2, mesothelin, and exogenous CD19). We also validated this in orthotopic xenograft models of glioblastoma and pancreatic cancer in vivo. CAR T cells exposed to wildtype (WT) or IFNgR1 KO tumor had similar transcriptional profiles, but the tumor cells had different signatures in response to CAR T cell co-culture. Further investigation showed marked upregulation of cell adhesion pathways in WT cells compared to IFNgR1 KO. We utilized microscopy with acoustic force and discovered CAR T cells had lower cell binding avidity to cells lacking IFNgR1 compared to WT cells. Following CAR T cell exposure, Intercellular Adhesion Molecule 1 (ICAM-1) was strongly upregulated at both the transcriptional and protein levels in WT cells but not IFNgR1 KO cells. We overexpressed ICAM-1 on IFNgR1 KO tumor cells and observed that CAR T cell binding avidity and cytotoxicity was restored to that of WT levels. This work highlights the importance of CAR T cell binding avidity and adhesion for optimal cytotoxicity. Better understanding of CAR T cell function will inform future construct design for successful therapies against solid tumors.
RCL was supported by T32 GM007306, T32 AI007529, and the Richard N. Cross Fund. ML was supported by T32 2T32CA071345-21A1. SRB was supported by T32CA009216-38. NJH was supported by the Landry Cancer Biology Fellowship. JJ is supported by a NIH F31 fellowship (1F31-MH117886). GG was partially funded by the Paul C. Zamecnik Chair in Oncology at the Massachusetts General Hospital Cancer Center and NIH R01CA 252940. MVM and this work is supported by the Damon Runyon Cancer Research Foundation, Stand Up to Cancer, NIH R01CA 252940, R01CA238268, and R01CA249062.
In 2014, the Centers for Disease Control and Prevention (CDC) launched “A Comprehensive Approach to Good Health and Wellness in Indian County” (GHWIC) to promote health and chronic disease prevention ...in tribal communities while facilitating cross-cultural learning and relationship-building. Through GHWIC, CDC aimed to work with American Indian and Alaska Native communities to identify effective health promotion strategies to address chronic disease disparities. Tribal sovereignty, community context, and consideration of tribal histories (e.g., oppression, genocide, and cultural erasure) are key to health improvement efforts and work with tribes. These elements center experience, knowledge, and self-determination to reclaim good health and wellness as Indigenous peoples see it. The Implementation Reflection Project was a qualitative inquiry composed of one-on-one discussions and small group sessions conducted to explore experiences of CDC staff, national partners, and tribal recipients as they implemented GHWIC program activities. The Project documented observations and recommendations for future tribal health funding efforts and identified best practices for effective partnerships with tribes and tribal organizations. Findings centered around tribal experiences with GHWIC, improved program processes, the importance of relationships, and the effects of internal capacity on implementation. Key suggestions for future work with tribal entities included simplifying and clarifying roles, expectations, and administration requirements, and establishing clear and consistent communication between program partners. The approach CDC used with GHWIC recipients was effective and respectful, but room for growth remains. Potential future collaborators in Indian Country should consider these findings when planning health promotion initiatives.
Approximately, 80,000 Americans are affected by sickle cell disease (SCD) and it is the most common inherited blood disorder in the United States. SCD is most prevalent among individuals of African, ...Mediterranean, Middle Eastern, and Asian Indian ancestry; however, statistics suggest that in the United States, African Americans are disproportionately affected by SCD (Sickle Cell Disease Association of America (SCDAA), 2007). Nearly, 1 in 400 African Americans is born with SCD; in addition, 1 in 12 African Americans is born with sickle cell trait (SCT) (National Human Genome Research Institute (NHGRI), 2007). Two carriers of the trait have a 25% chance of having an unaffected child, a 50% chance of having a child who is also a carrier, and a 25% chance of having a child with SCD. However, many African Americans lack knowledge about SCD and SCT and are unaware of their carrier status. The purpose of this two-phased sequential mixed methods study was to explore African American college students’ beliefs, attitudes, and knowledge of sickle cell disease (SCD) genetics, and of sickle cell trait (SCT) and SCD carrier testing. This purpose was accomplished by surveying 191 African American men and women between the ages of 19 and 30 years and then exploring those results in more depth through follow-up interviews with 8 purposefully selected individuals from the first phase. In the 1st phase, quantitative research questions addressed the relationship of African American college students’ current level of knowledge and attitude about SCD and SCT, attitude toward SCD carrier testing, and their intention to participate in carrier testing at various sites. A stepwise multiple regression analysis was used to determine which of these variables contributed most significantly to the participants’ decision to participate in carrier testing. In the 2nd phase, qualitative interviews with purposefully sampled individuals who participated in testing were used to explore the significance of participants’ attitude toward carrier testing. There have been few studies published that examined the knowledge and beliefs of young adults regarding SCD and SCD carrier testing. It was determined that the major factors that contributed to the students’ intention to participate in SCD carrier testing were attitude toward carrier testing, lack of knowledge regarding SCD and SCD carrier status, family history, cost, time and opportunity. It was revealed that the participants supported carrier testing and viewed SCD carrier testing for young adults as valuable. However, greater community health education about SCD and SCD carrier testing was desired. The implications for health education and promotion research are to (a) increase the SCD genetic knowledge of young adults in the African American community and (b) increase opportunities for minority populations to be informed of SCD carrier testing.
Thesis (Ph. D.)--University of Alabama at Birmingham, 2007.
Title from first page of PDF file (viewed Oct. 13, 2008). Includes bibliographical references (p. 174-183).
A 3D vertical seismic profiling (VSP) survey was acquired using a distributed acoustic sensing (DAS) system in the Permian Basin, West Texas. In total, 682 shot points from a pair of vibroseis units ...were recorded using optical fibers installed in a 9000 ft (2743 m) vertical part and 5000 ft (1524 m) horizontal reach of a well. Transmitted and reflected P, S, and converted waves were evident in the DAS data. From first-break P and S arrivals, we found average P-wave velocities of approximately 14,000 ft/s (4570 m/s) and S-wave velocities of 8800 ft/s (3000 m/s) in the deep section. We modified the conventional geophone VSP processing workflow and produced P-P reflection and P-S volumes derived from the well's vertical section. The Wolfcamp formation can be seen in two 3D volumes (P-P and P-S) from the vertical section of the well. They cover an area of 3000 ft (914 m) in the north-south direction and 1500 ft (460 m) in the west-east direction. Time slices showed coherent reflections, especially at 1.7 s (~11,000 ft), which was interpreted as the bottom of the Wolfcamp formation. Vp/Vs values from 2300 ft (701 m) -8800 ft (2682 m) interval range were between 1.7 and 2.0. These first data provide baseline images to compare to follow-up surveys after hydraulic fracturing as well as potential usefulness in extracting elastic properties and providing further indications of fractured volumes.
Near‐infrared (NIR) activatable upconversion nanoparticles (UCNPs) enable wireless‐based phototherapies by converting deep‐tissue‐penetrating NIR to visible light. UCNPs are therefore ideal as ...wireless transducers for photodynamic therapy (PDT) of deep‐sited tumors. However, the retention of unsequestered UCNPs in tissue with minimal options for removal limits their clinical translation. To address this shortcoming, biocompatible UCNPs implants are developed to deliver upconversion photonic properties in a flexible, optical guide design. To enhance its translatability, the UCNPs implant is constructed with an FDA‐approved poly(ethylene glycol) diacrylate (PEGDA) core clad with fluorinated ethylene propylene (FEP). The emission spectrum of the UCNPs implant can be tuned to overlap with the absorption spectra of the clinically relevant photosensitizer, 5‐aminolevulinic acid (5‐ALA). The UCNPs implant can wirelessly transmit upconverted visible light till 8 cm in length and in a bendable manner even when implanted underneath the skin or scalp. With this system, it is demonstrated that NIR‐based chronic PDT is achievable in an untethered and noninvasive manner in a mouse xenograft glioblastoma multiforme (GBM) model. It is postulated that such encapsulated UCNPs implants represent a translational shift for wireless deep‐tissue phototherapy by enabling sequestration of UCNPs without compromising wireless deep‐tissue light delivery.
Biocompatible upconversion hydrogel implants synergistically conserve the light‐guiding and near‐infrared transducing properties into a wireless light‐delivery implant. Such hydrogel‐based upconversion designs are explored to activate clinically relevant photosensitizer in wireless photodynamic therapy of glioblastoma multiforme. Upconversion implantable is potentially translatable to other phototherapies and as medical implants.
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