Development of a clinically relevant ovarian cancer model incorporating surgical cytoreduction to evaluate treatment of micro-metastatic disease.

OBJECTIVES: Mouse models of ovarian cancer commonly transfer large numbers of tumor cells into the peritoneal cavity to establish experimental metastatic disease, which may not adequately model early metastatic spread from a primary tumor site. We hypothesized we could develop an ovarian cancer model that predictably represents micro-metastatic disease. METHODS: Murine ID8VEGF ovarian cancer cells were transduced to express enhanced luciferase (eLuc) to enable intravital detection of microscopic disease burden and injected beneath the ovarian bursa of C57Bl/6 mice. At 6 or 10 weeks after orthotopic injection, when mice had detectable metastases, hysterectomy and bilateral salpingo-oophorectomy was performed to remove all macroscopic disease, and survival monitored. Immunohistochemistry and gene expression profiling were performed on primary and metastatic tumors. RESULTS: eLuc-transduced ID8VEGF cells were brighter than cells transduced with standard luciferase, enabling in vivo visualization of microscopic intra-abdominal metastases developing after orthotopic injection. Primary surgical cytoreduction removed the primary tumor mass but left minimal residual disease in all mice. Metastatic sites that developed following orthotopic injection were similar to metastatic human ovarian cancer sites. Gene expression and immune infiltration were similar between primary and metastatic mouse tumors. Surgical cytoreduction prolonged survival compared to no surgery, with earlier cytoreduction more beneficial than delayed, despite micro-metastatic disease in both settings. CONCLUSIONS: Mice with primary ovarian tumors established through orthotopic injection develop progressively fatal metastatic ovarian cancer, and benefit from surgical cytoreduction to remove bulky disease. This model enables the analysis of therapeutic regimens designed to target and potentially eradicate established minimal residual disease.

A CD200R-CD28 fusion protein appropriates an inhibitory signal to enhance T-cell function and therapy of murine leukemia.

Acute myeloid leukemia (AML), the most common adult acute leukemia in the United States, has the poorest survival rate, with 26% of patients surviving 5 years. Adoptive immunotherapy with T cells genetically modified to recognize tumors is a promising and evolving treatment option. However, antitumor activity, particularly in the context of progressive leukemia, can be dampened both by limited costimulation and triggering of immunoregulatory checkpoints that attenuate T-cell responses. Expression of CD200 (OX2), a negative regulator of T-cell function that binds CD200 receptor (CD200R), is commonly increased in leukemia and other malignancies and is associated with poor prognosis in leukemia patients. To appropriate and redirect the inhibitory effects of CD200R signaling on transferred CD8+ T cells, we engineered CD200R immunomodulatory fusion proteins (IFPs) with the cytoplasmic tail replaced by the signaling domain of the costimulatory receptor, CD28. An analysis of a panel of CD200R-CD28 IFP constructs revealed that the most effective costimulation was achieved in IFPs containing a dimerizing motif and a predicted tumor-T-cell distance that facilitates localization to the immunological synapse. T cells transduced with the optimized CD200R-CD28 IFPs exhibited enhanced proliferation and effector function in response to CD200+ leukemic cells in vitro. In adoptive therapy of disseminated leukemia, CD200R-CD28-transduced leukemia-specific CD8 T cells eradicated otherwise lethal disease more efficiently than wild-type cells and bypassed the requirement for interleukin-2 administration to sustain in vivo activity. The transduction of human primary T cells with the equivalent human IFPs increased proliferation and cytokine production in response to CD200+ leukemia cells, supporting clinical translation. This trial was registered at www.clinicaltrials.gov as #NCT01640301.

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.

A Fas-4-1BB fusion protein converts a death to a pro-survival signal and enhances T cell therapy.

Adoptive T cell therapy (ACT) with genetically modified T cells has shown impressive results against some hematologic cancers, but efficacy in solid tumors can be limited by restrictive tumor microenvironments (TMEs). For example, Fas ligand is commonly overexpressed in TMEs and induces apoptosis in tumor-infiltrating, Fas receptor-positive lymphocytes. We engineered immunomodulatory fusion proteins (IFPs) to enhance ACT efficacy, combining an inhibitory receptor ectodomain with a costimulatory endodomain to convert negative into positive signals. We developed a Fas-4-1BB IFP that replaces the Fas intracellular tail with costimulatory 4-1BB. Fas-4-1BB IFP-engineered murine T cells exhibited increased pro-survival signaling, proliferation, antitumor function, and altered metabolism in vitro. In vivo, Fas-4-1BB ACT eradicated leukemia and significantly improved survival in the aggressive KPC pancreatic cancer model. Fas-4-1BB IFP expression also enhanced primary human T cell function in vitro. Thus, Fas-4-1BB IFP expression is a novel strategy to improve multiple T cell functions and enhance ACT against solid tumors and hematologic malignancies.

Engineered Adoptive T-cell Therapy Prolongs Survival in a Preclinical Model of Advanced-Stage Ovarian Cancer.

Adoptive T-cell therapy using high-affinity T-cell receptors (TCR) to target tumor antigens has potential for improving outcomes in high-grade serous ovarian cancer (HGSOC) patients. Ovarian tumors develop a hostile, multicomponent tumor microenvironment containing suppressive cells, inhibitory ligands, and soluble factors that facilitate evasion of antitumor immune responses. Developing and validating an immunocompetent mouse model of metastatic ovarian cancer that shares antigenic and immunosuppressive qualities of human disease would facilitate establishing effective T-cell therapies. We used deep transcriptome profiling and IHC analysis of human HGSOC tumors and disseminated mouse ID8VEGF tumors to compare immunologic features. We then evaluated the ability of CD8 T cells engineered to express a high-affinity TCR specific for mesothelin, an ovarian cancer antigen, to infiltrate advanced ID8VEGF murine ovarian tumors and control tumor growth. Human CD8 T cells engineered to target mesothelin were also evaluated for ability to kill HLA-A2+ HGSOC lines. IHC and gene-expression profiling revealed striking similarities between tumors of both species, including processing/presentation of a leading candidate target antigen, suppressive immune cell infiltration, and expression of molecules that inhibit T-cell function. Engineered T cells targeting mesothelin infiltrated mouse tumors but became progressively dysfunctional and failed to persist. Treatment with repeated doses of T cells maintained functional activity, significantly prolonging survival of mice harboring late-stage disease at treatment onset. Human CD8 T cells engineered to target mesothelin were tumoricidal for three HGSOC lines. Treatment with engineered T cells may have clinical applicability in patients with advanced-stage HGSOC.