Differential modulation of cellular phenotype and drug sensitivity by extracellular matrix proteins in primary and metastatic pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is reported to be the third highest cause of cancer-related deaths in the United States. PDAC is known for its high proportion of stroma, which accounts for 90% of the tumor mass. The stroma is made up of extracellular matrix (ECM) and nonmalignant cells such as inflammatory cells, cancer-associated fibroblasts, and lymphatic and blood vessels. Here, we decoupled the effects of the ECM on PDAC cell lines by culturing cells on surfaces coated with different ECM proteins. Our data show that the primary tumor-derived cell lines have different morphology depending on the ECM proteins on which they are cultured, while metastatic lesion-derived PDAC lines' morphology does not change with respect to the different ECM proteins. Similarly, ECM proteins modulate the proliferation rate and the gemcitabine sensitivity of the primary tumor PDAC cell lines, but not the metastatic PDAC lines. Lastly, transcriptomics analysis of the primary tumor PDAC cells cultured on different ECM proteins reveals the regulation of various pathways, such as cell cycle, cell-adhesion molecules, and focal adhesion, including the regulation of several integrin genes that are essential for ECM recognition.

Suppressing or Enhancing Macrophage Engulfment through the Use of CD47 and Related Peptides.

Foreign particles and microbes are rapidly cleared by macrophages in vivo, although many key aspects of uptake mechanisms remain unclear. "Self" cells express CD47 which functions as an anti-phagocytic ligand for SIRPα on macrophages, particularly when pro-phagocytic ligands such as antibodies are displayed in parallel. Here, we review CD47 and related "Self" peptides as modulators of macrophage uptake. Nanoparticles conjugated with either CD47 or peptides derived from its SIRPα binding site can suppress phagocytic uptake by macrophages in vitro and in vivo, with similar findings for CD47-displaying viruses. Drugs, dyes, and genes as payloads thus show increased delivery to targeted cells. On the other hand, CD47 expression by cancer cells enables such cells to evade macrophages and immune surveillance. This has motivated development of soluble antagonists to CD47-SIRPα, ranging from blocking antibodies in the clinic to synthetic peptides in preclinical models. CD47 and peptides are thus emerging as dual-use phagocytosis modulators against diseases.

Human CD47-Derived Cyclic Peptides Enhance Engulfment of mAb-Targeted Melanoma by Primary Macrophages.

CD47 on healthy cells, cancer cells, and even engineered particles can inhibit phagocytic clearance by binding SIRPα on macrophages. To mimic and modulate this interaction with peptides that could be used as soluble antagonists or potentially as bioconjugates to surfaces, we made cyclic "nano-Self" peptides based on the key interaction loop of human CD47. Melanoma cells were studied as a standard preclinical cancer model and were antibody-opsonized to adhere to and activate engulfment by primary mouse macrophages. Phagocytosis in the presence of soluble peptides showed cyclic > wildtype > scrambled activity, with the same trend observed with human cells. Opsonized cells that were not engulfed adhered tightly to macrophages, with opposite trends to phagocytosis. Peptide activity is nonetheless higher in human versus mouse assays, consistent with species differences in CD47-SIRPα. Small peptides thus function as soluble antagonists of a major macrophage checkpoint.

Multivalent, Soluble Nano-Self Peptides Increase Phagocytosis of Antibody-Opsonized Targets while Suppressing "Self" Signaling.

Macrophages engulf "foreign" cells and particles, but phagocytosis of healthy cells and cancer cells is inhibited by expression of the ubiquitous membrane protein CD47 which binds SIRPα on macrophages to signal "self". Motivated by some clinical efficacy of anti-CD47 against liquid tumors and based on past studies of CD47-derived polypeptides on particles that inhibited phagocytosis of the particles, here we design soluble, multivalent peptides to bind and block SIRPα. Bivalent and tetravalent nano-Self peptides prove more potent (Keff ∼ 10 nM) than monovalent 8-mers as agonists for phagocytosis of antibody opsonized cells, including cancer cells. Multivalent peptides also outcompete soluble CD47 binding to human macrophages, consistent with SIRPα binding, and the peptides suppress phosphotyrosine in macrophages, consistent with inhibition of SIRPα's "self" signaling. Peptides exhibit minimal folding, but functionality suggests an induced fit into SIRPα's binding pocket. Pre-clinical studies in mice indicate safety, with no anemia that typifies clinical infusions of anti-CD47. Multivalent nano-Self peptides thus constitute an alternative approach to promoting phagocytosis of "self", including cancer cells targeted clinically.

Macrophage checkpoint blockade: results from initial clinical trials, binding analyses, and CD47-SIRPα structure-function.

The macrophage checkpoint is an anti-phagocytic interaction between signal regulatory protein alpha (SIRPα) on a macrophage and CD47 on all types of cells - ranging from blood cells to cancer cells. This interaction has emerged over the last decade as a potential co-target in cancer when combined with other anti-cancer agents, with antibodies against CD47 and SIRPα currently in preclinical and clinical development for a variety of hematological and solid malignancies. Monotherapy with CD47 blockade is ineffective in human clinical trials against many tumor types tested to date, except for rare cutaneous and peripheral lymphomas. In contrast, pre-clinical results show efficacy in multiple syngeneic mouse models of cancer, suggesting that many of these tumor models are more immunogenic and likely artificial compared to human tumors. However, combination therapies in humans of anti-CD47 with agents such as the anti-tumor antibody rituximab do show efficacy against liquid tumors (lymphoma) and are promising. Here, we review such trials as well as key interaction and structural features of CD47-SIRPα.