Tumor-resident regulatory T cells in pancreatic cancer express the αvß5 integrin as a targetable activation marker.

Pancreatic ductal adenocarcinoma (PDAC) has abundant immunosuppressive regulatory T cells (Tregs), which contribute to a microenvironment resistant to immunotherapy. Here, we report that Tregs in the PDAC tissue, but not those in the spleen, express the αvß5 integrin in addition to neuropilin-1 (NRP-1), which makes them susceptible to the iRGD tumor-penetrating peptide, which targets cells positive for αv integrin- and NRP-1. As a result, long-term treatment of PDAC mice with iRGD leads to tumor-specific depletion of Tregs and improved efficacy of immune checkpoint blockade. αvß5 integrin + Tregs are induced from both naïve CD4 + T cells and natural Tregs upon T cell receptor stimulation, and represent a highly immunosuppressive subpopulation of CCR8 + Tregs. This study identifies the αvß5 integrin as a marker for activated tumor-resident Tregs, which can be targeted to achieve tumor-specific Treg depletion and thereby augment anti-tumor immunity for PDAC therapy.

Syk Inhibition Reprograms Tumor-Associated Macrophages and Overcomes Gemcitabine-Induced Immunosuppression in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an insidious disease with a low 5-year survival rate. PDAC is characterized by infiltration of abundant tumor-associated macrophages (TAM), which promote immune tolerance and immunotherapeutic resistance. Here we report that macrophage spleen tyrosine kinase (Syk) promotes PDAC growth and metastasis. In orthotopic PDAC mouse models, genetic deletion of myeloid Syk reprogrammed macrophages into immunostimulatory phenotype, increased the infiltration, proliferation, and cytotoxicity of CD8+ T cells, and repressed PDAC growth and metastasis. Furthermore, gemcitabine (Gem) treatment induced an immunosuppressive microenvironment in PDAC by promoting protumorigenic polarization of macrophages. In contrast, treatment with the FDA-approved Syk inhibitor R788 (fostamatinib) remodeled the tumor immune microenvironment, "re-educated" protumorigenic macrophages towards an immunostimulatory phenotype and boosted CD8+ T-cell responses in Gem-treated PDAC in orthotopic mouse models and an ex vivo human pancreatic slice culture model. These findings illustrate the potential of Syk inhibition for enhancing the antitumor immune responses in PDAC and support the clinical evaluation of R788 either alone or together with Gem as a potential treatment strategy for PDAC. SIGNIFICANCE: Syk blockade induces macrophage polarization to an immunostimulatory phenotype, which enhances CD8+ T-cell responses and improves gemcitabine efficacy in pancreatic ductal adenocarcinoma, a clinically challenging malignancy.

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens.

Pseudomyxoma peritonei (PMP) is a rare condition that results from the dissemination of a mucinous primary tumor and the resultant accumulation of mucin-secreting tumor cells in the peritoneal cavity. PMP can arise from various types of cancers, including appendiceal, ovarian, and colorectal, though appendiceal neoplasms are by far the most common etiology. PMP is challenging to study due to its (1) rarity, (2) limited murine models, and (3) mucinous, acellular histology. The method presented here allows real-time visualization and interrogation of these tumor types using patient-derived ex vivo organotypic slices in a preparation where the tumor microenvironment (TME) remains intact. In this protocol, we first describe the preparation of tumor slices using a vibratome and subsequent long-term culture. Second, we describe confocal imaging of tumor slices and how to monitor functional readouts of viability, calcium imaging, and local proliferation. In short, slices are loaded with imaging dyes and are placed in an imaging chamber that can be mounted onto a confocal microscope. Time-lapse videos and confocal images are used to assess the initial viability and cellular functionality. This procedure also explores translational cellular movement, and paracrine signaling interactions in the TME. Lastly, we describe a dissociation protocol for tumor slices to be used for flow cytometry analysis. Quantitative flow cytometry analysis can be used for bench-to-bedside therapeutic testing to determine changes occurring within the immune landscape and epithelial cell content.

An Ex Vivo Organotypic Culture Platform for Functional Interrogation of Human Appendiceal Cancer Reveals a Prominent and Heterogenous Immunological Landscape.

PURPOSE: Epithelial neoplasms of the appendix are difficult to study preclinically given their low incidence, frequent mucinous histology, and absence of a comparable organ in mice for disease modeling. Although surgery is an effective treatment for localized disease, metastatic disease has a poor prognosis as existing therapeutics borrowed from colorectal cancer have limited efficacy. Recent studies reveal that appendiceal cancer has a genomic landscape distinct from colorectal cancer and thus preclinical models to study this disease are a significant unmet need. EXPERIMENTAL DESIGN: We adopted an ex vivo slice model that permits the study of cellular interactions within the tumor microenvironment. Mucinous carcinomatosis peritonei specimens obtained at surgical resection were cutoff using a vibratome to make 150-µm slices cultured in media. RESULTS: Slice cultures were viable and maintained their cellular composition regarding the proportion of epithelial, immune cells, and fibroblasts over 7 days. Within donor specimens, we identified a prominent and diverse immune landscape and calcium imaging confirmed that immune cells were functional for 7 days. Given the diverse immune landscape, we treated slices with TAK981, an inhibitor of SUMOylation with known immunomodulatory functions, in early-phase clinical trials. In 5 of 6 donor samples, TAK981-treated slices cultures had reduced viability, and regulatory T cells (Treg). These data were consistent with TAK981 activity in purified Tregs using an in vitro murine model. CONCLUSIONS: This study demonstrates an approach to study appendiceal cancer therapeutics and pathobiology in a preclinical setting. These methods may be broadly applicable to the study of other malignancies.

Using Organotypic Tissue Slices to Investigate the Microenvironment of Pancreatic Cancer: Pharmacotyping and Beyond.

Organotypic tissue slices prepared from patient tumors are a semi-intact ex vivo preparation that recapitulates many aspects of the tumor microenvironment (TME). While connections to the vasculature and nervous system are severed, the integral functional elements of the tumor remain intact for many days during the slice culture. During this window of time, the slice platforms offer a suite of molecular, biomechanical and functional tools to investigate PDAC biology. In this review, we first briefly discuss the development of pancreatic tissue slices as a model system. Next, we touch upon using slices as an orthogonal approach to study the TME as compared to other established 3D models, such as organoids. Distinct from most other models, the pancreatic slices contain autologous immune and other stromal cells. Taking advantage of the existing immune cells within the slices, we will discuss the breakthrough studies which investigate the immune compartment in the pancreas slices. These studies will provide an important framework for future investigations seeking to exploit or reprogram the TME for cancer therapy.

Tumor-penetrating therapy for ß5 integrin-rich pancreas cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by marked desmoplasia and drug resistance due, in part, to poor drug delivery to extravascular tumor tissue. Here, we report that carcinoma-associated fibroblasts (CAFs) induce ß5 integrin expression in tumor cells in a TGF-ß dependent manner, making them an efficient drug delivery target for the tumor-penetrating peptide iRGD. The capacity of iRGD to deliver conjugated and co-injected payloads is markedly suppressed when ß5 integrins are knocked out in the tumor cells. Of note, ß5 integrin knock-out in tumor cells leads to reduced disease burden and prolonged survival of the mice, demonstrating its contribution to PDAC progression. iRGD significantly potentiates co-injected chemotherapy in KPC mice with high ß5 integrin expression and may be a powerful strategy to target an aggressive PDAC subpopulation.

Antiapoptotic Role for Lifeguard in T Cell Mediated Immune Response.

Anti-apoptotic protein Lifeguard (LFG) is upregulated on T cells upon in vitro activation. To investigate its role in T cell immunity we infected wild type and LFG knockout bone marrow chimaeras mice with LCMV. We observed a decreased number of LFG KO activated CD8 and CD4 T cells throughout the infection and a marked decrease in LFG KO LCMV specific memory T cells. WT and KO T cells proliferated at the same rate, however, LFG KO CD44(hi) T cells showed increased cell death during the initial phase of the immune response. LFG KO and WT T cells were equally sensitive to the FAS antibody Jo-2 in ex vivo cultures, and blocking extrinsic pathways of cell death in vivo with Fas L or caspase 8 inhibitors did not rescue the increased apoptosis in LFG KO T cells. Our data suggest that LFG plays a role in T cell survival during the initial phase of anti-viral immune response by protecting pre-existing memory T cells and possibly newly activated T cells resulting in a diminished immune response and a decreased number of LCMV specific memory T cells.

Etchable plasmonic nanoparticle probes to image and quantify cellular internalization.

There is considerable interest in using nanoparticles as labels or to deliver drugs and other bioactive compounds to cells in vitro and in vivo. Fluorescent imaging, commonly used to study internalization and subcellular localization of nanoparticles, does not allow unequivocal distinction between cell surface-bound and internalized particles, as there is no methodology to turn particles 'off'. We have developed a simple technique to rapidly remove silver nanoparticles outside living cells, leaving only the internalized pool for imaging or quantification. The silver nanoparticle (AgNP) etching is based on the sensitivity of Ag to a hexacyanoferrate-thiosulphate redox-based destain solution. In demonstration of the technique we present a class of multicoloured plasmonic nanoprobes comprising dye-labelled AgNPs that are exceptionally bright and photostable, carry peptides as model targeting ligands, can be etched rapidly and with minimal toxicity in mice, and that show tumour uptake in vivo.

Censoring of self-reactive B cells by follicular dendritic cell-displayed self-antigen.

In the secondary lymphoid organs, intimate contact with follicular dendritic cells (FDCs) is required for B cell retention and Ag-driven selection during the germinal center response. However, selection of self-reactive B cells by Ag on FDCs has not been addressed. To this end, we generated a mouse model to conditionally express a membrane-bound self-antigen on FDCs and to monitor the fate of developing self-reactive B cells. In this article, we show that self-antigen displayed on FDCs mediates effective elimination of self-reactive B cells at the transitional stage. Notwithstanding, some self-reactive B cells persist beyond this checkpoint, showing evidence of Ag experience and intact proximal BCR signaling, but they are short-lived and unable to elicit T cell help. These results implicate FDCs as an important component of peripheral B cell tolerance that prevents the emergence of naive B cells capable of responding to sequestered self-antigens.

Antiapoptotic protein Lifeguard is required for survival and maintenance of Purkinje and granular cells.

Lifeguard (LFG) is an inhibitor of Fas-mediated cell death and is highly expressed in the cerebellum. We investigated the biological role of LFG in the cerebellum in vivo, using mice with reduced LFG expression generated by shRNA lentiviral transgenesis (shLFG mice) as well as LFG null mice. We found that LFG plays a role in cerebellar development by affecting cerebellar size, internal granular layer (IGL) thickness, and Purkinje cell (PC) development. All these features are more severe in early developmental stages and show substantial recovery overtime, providing a remarkable example of cerebellar plasticity. In adult mice, LFG plays a role in PC maintenance shown by reduced cellular density and abnormal morphology with increased active caspase 8 and caspase 3 immunostaining in shLFG and knockout (KO) PCs. We studied the mechanism of action of LFG as an inhibitor of the Fas pathway and provided evidence of the neuroprotective role of LFG in cerebellar granule neurons (CGNs) and PCs in an organotypic cerebellar culture system. Biochemical analysis of the Fas pathway revealed that LFG inhibits Fas-mediated cell death by interfering with caspase 8 activation. This result is supported by the increased number of active caspase 8-positive PCs in adult mice lacking LFG. These data demonstrate that LFG is required for proper development and survival of granular and Purkinje cells and suggest LFG may play a role in cerebellar disorders.

Organotypic cerebellar cultures: apoptotic challenges and detection.

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 (1,2) and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure (3,4). A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice. The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.