Heterogeneity of response to immune checkpoint blockade in hypermutated experimental gliomas

Intrinsic malignant brain tumors, such as glioblastomas are frequently resistant to immune checkpoint blockade (ICB) with few hypermutated glioblastomas showing response. Modeling patient-individual resistance is challenging due to the lack of predictive biomarkers and limited accessibility of tissue for serial biopsies. Here, we investigate resistance mechanisms to anti-PD-1 and anti-CTLA-4 therapy in syngeneic hypermutated experimental gliomas and show a clear dichotomy and acquired immune heterogeneity in ICB-responder and non-responder tumors. We made use of this dichotomy to establish a radiomic signature predicting tumor regression after pseudoprogression induced by ICB therapy based on serial magnetic resonance imaging. We provide evidence that macrophage-driven ICB resistance is established by CD4 T cell suppression and T$_{reg}$ expansion in the tumor microenvironment via the PD-L1/PD-1/CD80 axis. These findings uncover an unexpected heterogeneity of response to ICB in strictly syngeneic tumors and provide a rationale for targeting PD-L1-expressing tumor-associated macrophages to overcome resistance to ICB

Proteomics Approach Highlights Early Changes in Human Fibroblasts-Pancreatic Ductal Adenocarcinoma Cells Crosstal

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer mortality worldwide. Non-specific symptoms, lack of biomarkers in the early stages, and drug resistance due to the presence of a dense fibrous stroma all contribute to the poor outcome of this disease. The extracellular matrix secreted by activated fibroblasts contributes to the desmoplastic tumor microenvironment formation. Given the importance of fibroblast activation in PDAC pathology, it is critical to recognize the mechanisms involved in the transformation of normal fibroblasts in the early stages of tumorigenesis. To this aim, we first identified the proteins released from the pancreatic cancer cell line MIA-PaCa2 by proteomic analysis of their conditioned medium (CM). Second, normal fibroblasts were treated with MIA-PaCa2 CM for 24 h and 48 h and their proteostatic changes were detected by proteomics. Pathway analysis indicated that treated fibroblasts undergo changes compatible with the activation of migration, vasculogenesis, cellular homeostasis and metabolism of amino acids and reduced apoptosis. These biological activities are possibly regulated by ITGB3 and TGFB1/2 followed by SMAD3, STAT3 and BAG3 activation. In conclusion, this study sheds light on the crosstalk between PDAC cells and associated fibroblasts. Data are available via ProteomeXchange with identifier PXD030974

Development of an anti-BAG3 humanized antibody for treatment of pancreatic cancer

We have previously shown that secreted BAG3 is a potential target for the treatment of pancreatic ductal adenocarcinoma and that pancreatic tumor growth and metastatic dissemination can be reduced by treatment with an anti-BAG3 murine antibody. Here, we used complementarity-determining region (CDR) grafting to generate a humanized version of the anti-BAG3 antibody that may be further developed for possible clinical use. We show that the humanized anti-BAG3 antibody, named BAG3-H2L4, abrogates BAG3 binding to macrophages and subsequent release of IL-6. Furthermore, it specifically localizes into tumor tissues and significantly inhibits the growth of Mia PaCa-2 pancreatic cancer cell xenografts. We propose BAG3-H2L4 antibody as a potential clinical candidate for BAG3-targeted therapy in pancreatic cancer

Combined effect of anti-BAG3 and anti-PD-1 treatment on macrophage infiltrate, CD8(+) T cell number and tumour growth in pancreatic cancer

We read with great interest the article by Zhang et al 1 showing that CD8+ cell infiltration in pancreatic tumours can be enhanced by depletion of myeloid cells (CD11b+ macrophages and myeloid-derived suppressor cells) and that the depletion of CD11b+ cells resulted in decreased PD-L1 expression on cancer cells thus impairing the triggering of the inhibitory receptor PD-1 on T cells.1 Recruitment and activation of CD8+ lymphocytes in tumours are suppressed by mechanisms only partially understood and rescuing CD8+ cell infiltrate in tumours is one of the objectives of immunotherapies.1 2 Tumour-associated macrophages (TAMs) play a crucial role in the relation between tumour cells and their environment.3 Here, we confirm the interplay between macrophages and CD8+ cells in pancreatic cancer and identify a potential way to exploit this enhancing effect of anti-PD-1 treatment. Indeed, we show that reduction of macrophage infiltrate, through treatment with an anti-Bcl-2-Associated athanoGene 3 (BAG3) antibody,4 results in increased number of CD8+ cells in pancreatic tumours in a murine model

Pancreatic beta-cell specific BAG3 knockout results in chronic hyperinsulinemia inducing insulin resistance

Background: Insulin, secreted from pancreatic islets of Langerhans, is of critical importance in regulating glucose homeostasis. Defective insulin secretion and/or the inability of tissues to respond to insulin results in insulin resistance and to several metabolic and organ alterations. We have previously demonstrated that BAG3 regulates insulin secretion. Herein we explored the consequences of beta-cells specific BAG3 deficiency in an animal model. Methods: We generated a beta-cells specific BAG3 knockout mouse model. Glucose and insulin tolerance tests, proteomics, metabolomics, and immunohistochemical analysis were used to investigate the role of BAG3 in regulating insulin secretion and the effects of chronic exposure to excessive insulin release in vivo. Results: Beta-cells specific BAG3 knockout results in primary hyperinsulinism due to excessive insulin exocytosis finally leading to insulin resistance. We demonstrate that resistance is mainly muscle-dependent while the liver remains insulin sensitive. The chronically altered metabolic condition leads in time to histopathological alterations in different organs. We observe elevated glycogen and lipid accumulation in the liver reminiscent of non-alcoholic fatty liver disease as well as mesangial matrix expansion and thickening of the glomerular basement membrane, resembling the histology of chronic kidney disease. Conclusion: Altogether, this study shows that BAG3 plays a role in insulin secretion and provides a model for the study of hyperinsulinemia and insulin resistance