Cancer-Associated Fibroblast-Like Tumor Cells Remodel the Ewing Sarcoma Tumor Microenvironment.

PURPOSE: Despite limited genetic and histologic heterogeneity, Ewing sarcoma (EwS) tumor cells are transcriptionally heterogeneous and display varying degrees of mesenchymal lineage specification in vitro. In this study, we investigated if and how transcriptional heterogeneity of EwS cells contributes to heterogeneity of tumor phenotypes in vivo. EXPERIMENTAL DESIGN: Single-cell proteogenomic-sequencing of EwS cell lines was performed and integrated with patient tumor transcriptomic data. Cell subpopulations were isolated by FACS for assessment of gene expression and phenotype. Digital spatial profiling and human whole transcriptome analysis interrogated transcriptomic heterogeneity in EwS xenografts. Tumor cell subpopulations and matrix protein deposition were evaluated in xenografts and patient tumors using multiplex immunofluorescence staining. RESULTS: We identified CD73 as a biomarker of highly mesenchymal EwS cell subpopulations in tumor models and patient biopsies. CD73+ tumor cells displayed distinct transcriptional and phenotypic properties, including selective upregulation of genes that are repressed by EWS::FLI1, and increased migratory potential. CD73+ cells were distinguished in vitro and in vivo by increased expression of matrisomal genes and abundant deposition of extracellular matrix (ECM) proteins. In epithelial-derived malignancies, ECM is largely deposited by cancer-associated fibroblasts (CAF), and we thus labeled CD73+ EwS cells, CAF-like tumor cells. Marked heterogeneity of CD73+ EwS cell frequency and distribution was detected in tumors in situ, and CAF-like tumor cells and associated ECM were observed in peri-necrotic regions and invasive foci. CONCLUSIONS: EwS tumor cells can adopt CAF-like properties, and these distinct cell subpopulations contribute to tumor heterogeneity by remodeling the tumor microenvironment. See related commentary by Kuo and Amatruda, p. 5002.

Carcinoma-associated fibroblast-like tumor cells remodel the Ewing sarcoma tumor microenvironment.

Tumor heterogeneity is a major driver of cancer progression. In epithelial-derived malignancies, carcinoma-associated fibroblasts (CAFs) contribute to tumor heterogeneity by depositing extracellular matrix (ECM) proteins that dynamically remodel the tumor microenvironment (TME). Ewing sarcomas (EwS) are histologically monomorphous, mesenchyme-derived tumors that are devoid of CAFs. Here we identify a previously uncharacterized subpopulation of transcriptionally distinct EwS tumor cells that deposit pro-tumorigenic ECM. Single cell analyses revealed that these CAF-like cells differ from bulk EwS cells by their upregulation of a matrisome-rich gene signature that is normally repressed by EWS::FLI1, the oncogenic fusion transcription factor that underlies EwS pathogenesis. Further, our studies showed that ECM-depositing tumor cells express the cell surface marker CD73, allowing for their isolation ex vivo and detection in situ. Spatial profiling of tumor xenografts and patient biopsies demonstrated that CD73 + EwS cells and tumor cell-derived ECM are prevalent along tumor borders and invasive fronts. Importantly, despite loss of EWS::FLI1-mediated gene repression, CD73 + EwS cells retain expression of EWS::FLI1 and the fusion-activated gene signature, as well as tumorigenic and proliferative capacities. Thus, EwS tumor cells can be reprogrammed to adopt CAF-like properties and these transcriptionally and phenotypically distinct cell subpopulations contribute to tumor heterogeneity by remodeling the TME.

FGFR4-Targeted Chimeric Antigen Receptors Combined with Anti-Myeloid Polypharmacy Effectively Treat Orthotopic Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue cancer in children. Treatment outcomes, particularly for relapsed/refractory or metastatic disease, have not improved in decades. The current lack of novel therapies and low tumor mutational burden suggest that chimeric antigen receptor (CAR) T-cell therapy could be a promising approach to treating RMS. Previous work identified FGF receptor 4 (FGFR4, CD334) as being specifically upregulated in RMS, making it a candidate target for CAR T cells. We tested the feasibility of an FGFR4-targeted CAR for treating RMS using an NSG mouse with RH30 orthotopic (intramuscular) tumors. The first barrier we noted was that RMS tumors produce a collagen-rich stroma, replete with immunosuppressive myeloid cells, when T-cell therapy is initiated. This stromal response is not seen in tumor-only xenografts. When scFV-based binders were selected from phage display, CARs targeting FGFR4 were not effective until our screening approach was refined to identify binders to the membrane-proximal domain of FGFR4. Having improved the CAR, we devised a pharmacologic strategy to augment CAR T-cell activity by inhibiting the myeloid component of the T-cell-induced tumor stroma. The combined treatment of mice with anti-myeloid polypharmacy (targeting CSF1R, IDO1, iNOS, TGFbeta, PDL1, MIF, and myeloid misdifferentiation) allowed FGFR4 CAR T cells to successfully clear orthotopic RMS tumors, demonstrating that RMS tumors, even with very low copy-number targets, can be targeted by CAR T cells upon reversal of an immunosuppressive microenvironment.

Genetically engineered macrophages persist in solid tumors and locally deliver therapeutic proteins to activate immune responses.

BACKGROUND: Though currently approved immunotherapies, including chimeric antigen receptor T cells and checkpoint blockade antibodies, have been successfully used to treat hematological and some solid tumor cancers, many solid tumors remain resistant to these modes of treatment. In solid tumors, the development of effective antitumor immune responses is hampered by restricted immune cell infiltration and an immunosuppressive tumor microenvironment (TME). An immunotherapy that infiltrates and persists in the solid TME, while providing local, stable levels of therapeutic to activate or reinvigorate antitumor immunity could overcome these challenges faced by current immunotherapies. METHODS: Using lentivirus-driven engineering, we programmed human and murine macrophages to express therapeutic payloads, including Interleukin (IL)-12. In vitro coculture studies were used to evaluate the effect of genetically engineered macrophages (GEMs) secreting IL-12 on T cells and on the GEMs themselves. The effects of IL-12 GEMs on gene expression profiles within the TME and tumor burden were evaluated in syngeneic mouse models of glioblastoma and melanoma and in human tumor slices isolated from patients with advanced gastrointestinal malignancies. RESULTS: Here, we present a cellular immunotherapy platform using lentivirus-driven genetic engineering of human and mouse macrophages to constitutively express proteins, including secreted cytokines and full-length checkpoint antibodies, as well as cytoplasmic and surface proteins that overcomes these barriers. GEMs traffic to, persist in, and express lentiviral payloads in xenograft mouse models of glioblastoma, and express a non-signaling truncated CD19 surface protein for elimination. IL-12-secreting GEMs activated T cells and induced interferon-gamma (IFNγ) in vitro and slowed tumor growth resulting in extended survival in vivo. In a syngeneic glioblastoma model, IFNγ signaling cascades were also observed in mice treated with mouse bone-marrow-derived GEMs secreting murine IL-12. These findings were reproduced in ex vivo tumor slices comprised of intact MEs. In this setting, IL-12 GEMs induced tumor cell death, chemokines and IFNγ-stimulated genes and proteins. CONCLUSIONS: Our data demonstrate that GEMs can precisely deliver titratable doses of therapeutic proteins to the TME to improve safety, tissue penetrance, targeted delivery and pharmacokinetics.

Characterization of the immune microenvironment of diffuse intrinsic pontine glioma: implications for development of immunotherapy.

Background: Diffuse intrinsic pontine glioma (DIPG) is a uniformly fatal CNS tumor diagnosed in 300 American children per year. Radiation is the only effective treatment and extends overall survival to a median of 11 months. Due to its location in the brainstem, DIPG cannot be surgically resected. Immunotherapy has the ability to target tumor cells specifically; however, little is known about the tumor microenvironment in DIPGs. We sought to characterize infiltrating immune cells and immunosuppressive factor expression in pediatric low- and high-grade gliomas and DIPG. Methods: Tumor microarrays were stained for infiltrating immune cells. RNA was isolated from snap-frozen tumor tissue and Nanostring analysis performed. DIPG and glioblastoma cells were co-cultured with healthy donor macrophages, T cells, or natural killer (NK) cells, and flow cytometry and cytotoxicity assays performed to characterize the phenotype and function, respectively, of the immune cells. Results: DIPG tumors do not have increased macrophage or T-cell infiltration relative to nontumor control, nor do they overexpress immunosuppressive factors such as programmed death ligand 1 and/or transforming growth factor ß1. H3.3-K27M DIPG cells do not repolarize macrophages, but are not effectively targeted by activated allogeneic T cells. NK cells lysed all DIPG cultures. Conclusions: DIPG tumors have neither a highly immunosuppressive nor inflammatory microenvironment. Therefore, major considerations for the development of immunotherapy will be the recruitment, activation, and retention of tumor-specific effector immune cells.

EGFR806-CAR T cells selectively target a tumor-restricted EGFR epitope in glioblastoma.

Targeting solid tumor antigens with chimeric antigen receptor (CAR) T cell therapy requires tumor specificity and tolerance toward variability in antigen expression levels. Given the relative paucity of unique cell surface proteins on tumor cells for CAR targeting, we have focused on identifying tumor-specific epitopes that arise as a consequence of target protein posttranslational modification. We designed a CAR using a mAb806-based binder, which recognizes tumor-specific untethered EGFR. The mAb806 epitope is also exposed in the EGFRvIII variant transcript. By varying spacer domain elements of the CAR, we structurally tuned the CAR to recognize low densities of EGFR representative of non-gene amplified expression levels in solid tumors. The appropriately tuned short-spacer 2nd generation EGFR806-CAR T cells showed efficient in vitro cytokine secretion and glioma cell lysis, which was competitively blocked by a short peptide encompassing the mAb806 binding site. Unlike the nonselective Erbitux-based CAR, EGFR806-CAR T cells did not target primary human fetal brain astrocytes expressing wild-type EGFR, but showed a similar level of activity compared to Erbitux-CAR when the tumor-specific EGFRvIII transcript variant was overexpressed in astrocytes. EGFR806-CAR T cells successfully treated orthotopic U87 glioma implants in NSG mice, with 50% of animals surviving to 90 days. With additional IL-2 support, all tumors were eradicate without recurrence after 90 days. In a novel human induced pluripotent stem cell (iPSC)-derived teratoma xenograft model, EGFR806-CAR T cells infiltrated but were not activated in EGFR+ epidermal cell nests as assessed by Granzyme B expression. These results indicate that EGFR806-CAR T cells effectively and selectively target EGFR-expressing tumor cells.

Effects of tumor grade and dexamethasone on myeloid cells in patients with glioma.

Efforts to reduce immunosuppression in the solid tumor microenvironment by blocking the recruitment or polarization of tumor associated macrophages (TAM), or myeloid derived suppressor cells (MDSCs), have gained momentum in recent years. Expanding our knowledge of the immune cell types, cytokines, or recruitment factors that are associated with high-grade disease, both within the tumor and in circulation, is critical to identifying novel targets for immunotherapy. Furthermore, a better understanding of how therapeutic regimens, such as Dexamethasone (Dex), chemotherapy, and radiation, impact these factors will facilitate the design of therapies that can be targeted to the appropriate populations and retain efficacy when administered in combination with standard of care regimens. Here we perform quantitative analysis of tissue microarrays made of samples taken from grades I-III astrocytoma and glioblastoma (GBM, grade IV astrocytoma) to evaluate infiltration of myeloid markers CD163, CD68, CD33, and S100A9. Serum, flow cytometric, and Nanostring analysis allowed us to further elucidate the impact of Dex treatment on systemic biomarkers, circulating cells, and functional markers within tumor tissue. We found that common myeloid markers were elevated in Dex-treated grade I astrocytoma and GBM compared to non-neoplastic brain tissue and grade II-III astrocytomas. Cell frequencies in these samples differed significantly from those in Dex-naïve patients in a pattern that depended on tumor grade. In contrast, observed changes in serum chemokines or circulating monocytes were independent of disease state and were due to Dex treatment alone. Furthermore, these changes seen in blood were often not reflected within the tumor tissue. Conclusions: Our findings highlight the importance of considering perioperative treatment as well as disease grade when assessing novel therapeutic targets or biomarkers of disease.

Chimeric Antigen Receptor T Cell-Mediated Neurotoxicity in Nonhuman Primates.

Chimeric antigen receptor (CAR) T-cell immunotherapy has revolutionized the treatment of refractory leukemias and lymphomas, but is associated with significant toxicities, namely cytokine release syndrome (CRS) and neurotoxicity. A major barrier to developing therapeutics to prevent CAR T cell-mediated neurotoxicity is the lack of clinically relevant models. Accordingly, we developed a rhesus macaque (RM) model of neurotoxicity via adoptive transfer of autologous CD20-specific CAR T cells. Following cyclophosphamide lymphodepletion, CD20 CAR T cells expand to 272 to 4,450 cells/µL after 7 to 8 days and elicit CRS and neurotoxicity. Toxicities are associated with elevated serum IL6, IL8, IL1RA, MIG, and I-TAC levels, and disproportionately high cerebrospinal fluid (CSF) IL6, IL2, GM-CSF, and VEGF levels. During neurotoxicity, both CD20 CAR and non-CAR T cells accumulate in the CSF and in the brain parenchyma. This RM model demonstrates that CAR T cell-mediated neurotoxicity is associated with proinflammatory CSF cytokines and a pan-T cell encephalitis.Significance: We provide the first immunologically relevant, nonhuman primate model of B cell-directed CAR T-cell therapy-mediated CRS and neurotoxicity. We demonstrate CAR and non-CAR T-cell infiltration in the CSF and in the brain during neurotoxicity resulting in pan-encephalitis, accompanied by increased levels of proinflammatory cytokines in the CSF. Cancer Discov; 8(6); 750-63. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 663.

Intent-to-treat leukemia remission by CD19 CAR T cells of defined formulation and dose in children and young adults.

Transitioning CD19-directed chimeric antigen receptor (CAR) T cells from early-phase trials in relapsed patients to a viable therapeutic approach with predictable efficacy and low toxicity for broad application among patients with high unmet need is currently complicated by product heterogeneity resulting from transduction of undefined T-cell mixtures, variability of transgene expression, and terminal differentiation of cells at the end of culture. A phase 1 trial of 45 children and young adults with relapsed or refractory B-lineage acute lymphoblastic leukemia was conducted using a CD19 CAR product of defined CD4/CD8 composition, uniform CAR expression, and limited effector differentiation. Products meeting all defined specifications occurred in 93% of enrolled patients. The maximum tolerated dose was 106 CAR T cells per kg, and there were no deaths or instances of cerebral edema attributable to product toxicity. The overall intent-to-treat minimal residual disease-negative (MRD-) remission rate for this phase 1 study was 89%. The MRD- remission rate was 93% in patients who received a CAR T-cell product and 100% in the subset of patients who received fludarabine and cyclophosphamide lymphodepletion. Twenty-three percent of patients developed reversible severe cytokine release syndrome and/or reversible severe neurotoxicity. These data demonstrate that manufacturing a defined-composition CD19 CAR T cell identifies an optimal cell dose with highly potent antitumor activity and a tolerable adverse effect profile in a cohort of patients with an otherwise poor prognosis. This trial was registered at www.clinicaltrials.gov as #NCT02028455.

Genetically Engineered Macrophages: A Potential Platform for Cancer Immunotherapy.

In spite of their successes against hematologic malignancies, immunotherapeutic interventions for the treatment of patients with glioblastoma (GBM) have thus far been unsuccessful. This is in part due to the presence of a tumor microenvironment that fosters neoplastic growth and protects the tumor from destruction by the immune system. A novel genetically engineered macrophage-based platform has been developed with the potential to minimize the effects of the suppressive tumor microenvironment and improve innate and adaptive antitumor immune responses. A newly described lentiviral expression system was validated for the generation of transduced monocytes and monocyte-derived macrophages, and transgene expression was shown to be stable over the course of weeks to months, both in vitro and in a mouse xenograft model of GBM. Furthermore, the genetically engineered macrophages (GEMs) neither caused morbidity in animals nor contributed to accelerated tumor growth. The versatility of GEMs is also highlighted by showing that they can be engineered to secrete proteins that either reduce immune suppression, such as the soluble transforming growth factor beta receptor II, or promote immune cell activation, by expressing interleukin 21. There is also the potential to prevent GEM-mediated immune suppression by using the CRISPR system to knock out genes responsible for dysfunction of cytotoxic cells, including interleukin 10 and programmed death-ligand 1. Together, these results suggest that GEMs are an ideal cell type for transforming the tumor microenvironment and enhancing antitumor immunity. Importantly, it is anticipated that these findings will have broad applicability to other types of tumors with microenvironments that currently preclude successful immunotherapeutic approaches.

NKG2D ligand expression in pediatric brain tumors.

Adult brain tumors establish an immunosuppressive tumor microenvironment as a modality of immune escape, with several immunotherapies designed to overcome this barrier. However, the relationship between tumor cells and immune cells in pediatric brain tumor patients is not as well-defined. In this study, we sought to determine whether the model of immune escape observed in adult brain tumors is reflected in patients with pediatric brain tumors by evaluating NKG2D ligand expression on tissue microarrays created from patients with a variety of childhood brain tumor diagnoses, and infiltration of Natural Killer and myeloid cells. We noted a disparity between mRNA and protein expression for the 8 known NKG2D ligands. Surprisingly, high-grade gliomas did not have increased NKG2D ligand expression compared to normal adjacent brain tissue, nor did they have significant myeloid or NK cell infiltration. These data suggest that pediatric brain tumors have reduced NK cell-mediated immune surveillance, and a less immunosuppressive tumor microenvironment as compared to their adult counterparts. These data indicate that therapies aimed to improve NK cell trafficking and functions in pediatric brain tumors may have a greater impact on anti-tumor immune responses and patient survival, with fewer obstacles to overcome.

Functional Tuning of CARs Reveals Signaling Threshold above Which CD8+ CTL Antitumor Potency Is Attenuated due to Cell Fas-FasL-Dependent AICD.

Chimeric antigen receptor (CAR) development is biased toward selecting constructs that elicit the highest magnitude of T-cell functional outputs. Here, we show that components of CAR extracellular spacer and cytoplasmic signaling domain modulate, in a cooperative manner, the magnitude of CD8(+)CTL activation for tumor-cell cytolysis and cytokine secretion. Unexpectedly, CAR constructs that generate the highest in vitro activity, either by extracellular spacer length tuning or by the addition of cytoplasmic signaling modules, exhibit attenuated antitumor potency in vivo, whereas CARs tuned for moderate signaling outputs mediate tumor eradication. Recursive CAR triggering renders CTLs expressing hyperactive CARs highly susceptible to activation-induced cell death (AICD) as a result of augmented FasL expression. CAR tuning using combinations of extracellular spacers and cytoplasmic signaling modules, which limit AICD of CD8(+)CTLs, may be a critical parameter for achieving clinical activity against solid tumors.

The adventitia: a progenitor cell niche for the vessel wall.

Recent observations suggest that the adventitial layer of blood vessels exhibits properties resembling a stem/progenitor cell niche. Progenitor cells have been isolated from the adventitia of both murine and human blood vessels with the potential to form endothelial cells, mural cells, osteogenic cells, and adipocytes. These progenitors appear to cluster at or near the border zone between the outer media and inner adventitia. In the mouse, this border zone region corresponds to a localized site of sonic hedgehog signaling in the artery wall. This brief review will discuss the emerging evidence that the tunica adventitia may provide a niche-like signaling environment for resident progenitor cells and will address the role of the adventitia in growth, remodeling, and repair of the artery wall.

The adventitia: a dynamic interface containing resident progenitor cells.

Conventional views of the tunica adventitia as a poorly organized layer of vessel wall composed of fibroblasts, connective tissue, and perivascular nerves are undergoing revision. Recent studies suggest that the adventitia has properties of a stem/progenitor cell niche in the artery wall that may be poised to respond to arterial injury. It is also a major site of immune surveillance and inflammatory cell trafficking and harbors a dynamic microvasculature, the vasa vasorum, that maintains the medial layer and provides an important gateway for macrophage and leukocyte migration into the intima. In addition, the adventitia is in contact with tissue that surrounds the vessel and may actively participate in exchange of signals and cells between the vessel wall and the tissue in which it resides. This brief review highlights recent advances in our understanding of the adventitia and its resident progenitor cells and discusses progress toward an integrated view of adventitial function in vascular development, repair, and disease.

Vascular smooth muscle progenitor cells: building and repairing blood vessels.

Molecular pathways that control the specification, migration, and number of available smooth muscle progenitor cells play key roles in determining blood vessel size and structure, capacity for tissue repair, and progression of age-related disorders. Defects in these pathways produce malformations of developing blood vessels, depletion of smooth muscle progenitor cell pools for vessel wall maintenance and repair, and aberrant activation of alternative differentiation pathways in vascular disease. A better understanding of the molecular mechanisms that uniquely specify and maintain vascular smooth muscle cell precursors is essential if we are to use advances in stem and progenitor cell biology and somatic cell reprogramming for applications directed to the vessel wall.