Adaptive drug-resistance mechanisms allow human tumors to evade treatment through selection and expansion of treatment-resistant clones. Here, studying clonal evolution of tumor cells derived from human pancreatic tumors, we demonstrate that in vitro cultures and in vivo tumors are maintained by a common set of tumorigenic cells that can be used to establish clonal replica tumors (CRTs), large cohorts of animals bearing human tumors with identical clonal composition. Using CRTs to conduct quantitative assessments of adaptive responses to therapeutics, we uncovered a multitude of functionally heterogeneous subpopulations of cells with differential degrees of drug sensitivity. High-throughput isolation and deep characterization of unique clonal lineages showed genetic and transcriptomic diversity underlying functionally diverse subpopulations. Molecular annotation of gemcitabine-naive clonal lineages with distinct responses to treatment in the context of CRTs generated signatures that can predict the response to chemotherapy, representing a potential biomarker to stratify patients with pancreatic cancer.
Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.

Donor T lymphocytes are directly responsible for graft-versus-host disease. Molecules important in T-cell function may, therefore, be appropriate targets for graft-versus-host disease therapy and/or prophylaxis. Here we analyzed whether nuclear factor-κ B inducing kinase might have a role in graft-versus-host disease.
We studied the expression of nuclear factor-κ B inducing kinase in human samples from patients with graft-versus-host disease. We also explored the effect of nuclear factor-κ B inducing kinase in a murine model of graft-versus-host disease using donor cells from aly/aly mice (deficient in nuclear factor-κ B inducing kinase) and C57BL/6 mice (control).
We detected expression of nuclear factor-κ B inducing kinase in T-lymphocytes in the pathological lesions of patients with acute graft-versus-host disease. Mice transplanted with aly/aly T lymphocytes did not develop graft-versus-host disease at all, while mice receiving C57BL/6 cells died of a lethal form of the disease. Deficiency of nuclear factor-κ B inducing kinase did not affect the engrafting ability of donor T cells, but severely impaired their expansion capacity early after transplantation, and aly/aly T cells showed a higher proportion of apoptosis than did C57BL/6 T cells. Effector T lymphocytes were the T-cell subset most affected by nuclear factor-κ B inducing kinase deficiency. We also detected lower amounts of inflammatory cytokines in the serum of mice receiving aly/aly T cells than in the serum of mice receiving C57BL/6 T cells.
Our results show that nuclear factor-κ B inducing kinase has a role in graft-versus-host disease by maintaining the viability of activated alloreactive T lymphocytes.

The GD2 ganglioside expressed on neuroectodermal tumor cells has been used as a target for passive and active immunotherapy in patients with malignant melanoma and neuroblastoma. We have reported that immunization of mice with a 47-LDA mimotope of GD2, isolated from a phage display peptide library with anti-GD2 mAb 14G2a, induces MHC class I-restricted CD8(+) T cell responses to syngeneic neuroblastoma tumor cells. The cytotoxic activity of the vaccine-induced CTLs was independent of GD2 expression, suggesting recognition of a novel tumor-associated Ag cross-reacting with 47-LDA. Glycan microarray and immunoblotting studies using 14G2a mAb demonstrated that this Ab is highly specific for the entire carbohydrate motif of GD2 but also cross-reacts with a 105 kDa glycoprotein expressed by GD2(+) and GD2(-) neuroblastoma and melanoma cells. Functional studies of tumor cells grown in three-dimensional collagen cultures with 14G2a mAb showed decreases in matrix metalloproteinase-2 activation, a process regulated by the 105 kDa-activated leukocyte cell adhesion molecule (ALCAM/CD166). A recombinant CD166 glycoprotein was shown to be recognized by 14G2a Ab and inhibition of CD166 expression by RNA interference ablated the cell sensitivity to lysis by 47-LDA-induced CD8(+) T cells in vitro and in vivo. The binding of 14G2a to CD166 was not disruptable by a variety of exo- and endo-glycosidases, implying recognition of a non-glycan epitope on CD166. These results suggest that the vaccine-induced CTLs recognize a 47-LDA cross-reactive epitope expressed by CD166, and reveal a novel mechanism of induction of potent tumor-specific cellular responses by mimotopes of tumor-associated carbohydrate Ags.

Exosomes are small membrane vesicles that are released into the extracellular environment during fusion of multivesicular bodies with plasma membrane. Exosomes are secreted by various cell types including hematopoietic cells, normal epithelial cells and even some tumor cells. They are known to carry MHC class I, various costimulatory molecules and some tetraspanins. Recent studies have shown the potential of using native exosomes as immunologic stimulants. Here, we demonstrate a novel means of using exosomes engineered to express a specific tumor antigen to generate an immune response against tumors. We expressed a target tumor antigen, human MUC1 (hMUC1), in 2 MHC type-distinct mouse cell lines, CT26 and TA3HA. Analysis of exosomes purified from these cells revealed that exosomes contained the target MUC1 antigen on their surfaces as well as other well-described exosomal proteins, including Hsc70 and MHC class I molecules. In addition, both autologous and allogenic exosomes were able to stimulate the activation of immune cells and suppress hMUC1-expressing tumor growth in a MUC1-specific and dose-related manner. Therefore, these data suggest that exosomes can be engineered from tumor cell lines to deliver a target immunogen capable of inducing an effective immune response and that they may represent a new cell-free tumor vaccine.

The oncofetal antigen immature laminin receptor protein (OFA-iLRP) is a highly conserved protein that is preferentially expressed in fetal tissues and in many types of cancer, including hematopoietic malignancies, whereas OFA-iLRP is not detectable on healthy differentiated adult cells. To investigate whether OFA-iLRP-specific cytotoxic T lymphocytes (CTLs) are capable of killing OFA-iLRP-expressing hematologic targets, CTLs were generated from healthy HLA-A*0201-positive volunteers by incubating T cells with autologous dendritic cells (DCs) transfected with OFA-iLRP RNA. OFA-iLRP-specific CTLs lysed HLA-A2+ OFA-iLRP+ tumor cells, including several lymphoma and leukemia cell lines, as well as fresh leukemic targets from patients with acute myeloid leukemia (AML) and chronic lymphatic leukemia (CLL), indicating that OFA-iLRP-derived peptides are naturally processed and presented by hematologic tumors. Healthy OFA-iLRP-negative target cells (CD14+ monocytes, activated B cells, DCs, bone marrow cells) were not attacked by OFA-iLRP-specific CTLs. Furthermore, in an established murine B-cell lymphoma model (A20), treatment with syngeneic DCs transfected with OFA-iLRP-coding RNA resulted in powerful antitumor effects in a significant portion of mice. For the first time, these data show that OFA-iLRP can be used as a target for T-cell-based immunotherapeutic strategies against hematologic malignancies.