Cory Alvey

Engineering macrophages to eat cancer: from “marker of self” CD47 and phagocytosis to differentiation

The ability of a macrophage to engulf and break down invading cells and other targets provides a first line of immune defense in nearly all tissues. This defining ability to “phagos” or devour can subsequently activate the entire immune system against foreign and diseased cells, and progress is now being made on a decades‐old idea of directing macrophages to phagocytose specific targets, such as cancer cells. Engineered T cells provide precedence with recent clinical successes against liquid tumors, but solid tumors remain a challenge, and a handful of clinical trials seek to exploit the abundance of tumor‐associated macrophages instead. Although macrophage differentiation into such phenotypes with deficiencies in phagocytic ability can raise challenges, newly recognized features of cancer cells that might be manipulated to increase the phagocytosis of those cells include ≥1 membrane protein, CD47, which broadly inhibits phagocytosis and is abundantly expressed on all healthy cells. Physical properties of the target also influence phagocytosis and again relate—via cytoskeleton forces—to differentiation pathways in solid tumors. Such pathways extend to mechanosensing by the nuclear lamina, which is known to influence signaling by soluble retinoids that can regulate the macrophage SIRPα, the receptor for CD47. Here, we highlight some of those past, present, and rapidly emerging efforts to understand and control macrophages for cancer therapy.

“Marker of Self” CD47 on lentiviral vectors decreases macrophage-mediated clearance and increases delivery to SIRPA-expressing lung carcinoma tumors

Lentiviruses infect many cell types and are now widely used for gene delivery in vitro, but in vivo uptake of these foreign vectors by macrophages is a limitation. Lentivectors are produced here from packaging cells that overexpress “Marker of Self” CD47, which inhibits macrophage uptake of cells when prophagocytic factors are also displayed. Single particle analyses show “hCD47-Lenti” display properly oriented human-CD47 for interactions with the macrophage’s inhibitory receptor SIRPA. Macrophages derived from human and NOD/SCID/Il2rg−/− (NSG) mice show a SIRPA-dependent decrease in transduction, i.e., transgene expression, by hCD47-Lenti compared to control Lenti. Consistent with known “Self” signaling pathways, macrophage transduction by control Lenti is decreased by drug inhibition of Myosin-II to the same levels as hCD47-Lenti. In contrast, human lung carcinoma cells express SIRPA and use it to enhance transduction by hCD47-Lenti- as illustrated by more efficient gene deletion using CRISPR/Cas9. Intravenous injection of hCD47-Lenti into NSG mice shows hCD47 prolongs circulation, unless a blocking anti-SIRPA is preinjected. In vivo transduction of spleen and liver macrophages also decreases for hCD47-Lenti while transduction of lung carcinoma xenografts increases. hCD47 could be useful when macrophage uptake is limiting on other viral vectors that are emerging in cancer treatments (e.g., Measles glycoprotein-pseudotyped lentivectors) and also in targeting various SIRPA-expressing tumors such as glioblastomas.

Membrane fluctuations and acidosis regulate cooperative binding of “marker of self” CD47 with macrophage checkpoint receptor SIRPα

ABSTRACT Cell-cell interactions that result from membrane proteins binding weakly in trans can cause accumulations in cis that suggest cooperativity and thereby an acute sensitivity to environmental factors. The ubiquitous ‘marker of self’ protein CD47 binds weakly to SIRPα on macrophages, which leads to accumulation of SIRPα (also known as SHPS-1, CD172A and SIRPA) at phagocytic synapses and ultimately to inhibition of engulfment of ‘self’ cells – including cancer cells. We reconstituted this macrophage checkpoint with GFP-tagged CD47 on giant vesicles generated from plasma membranes and then imaged vesicles adhering to SIRPα immobilized on a surface. CD47 diffusion is impeded near the surface, and the binding-unbinding events reveal cooperative interactions as a concentration-dependent two-dimensional affinity. Membrane fluctuations out-of-plane link cooperativity to membrane flexibility with suppressed fluctuations in the vicinity of bound complexes. Slight acidity (pH 6) stiffens membranes, diminishes cooperative interactions and also reduces ‘self’ signaling of cancer cells in phagocytosis. Sensitivity of cell-cell interactions to microenvironmental factors – such as the acidity of tumors and other diseased or inflamed sites – can thus arise from the collective cooperative properties of flexible membranes. This article has an associated First Person interview with the first author of the paper. Summary: Binding of membrane proteins smooths the fluctuating membrane and facilitates more binding, which can be very important for weak immunological interactions such as the interaction of macrophage SIRPα with CD47 on cancer cells.

Nuclear rupture at sites of high curvature compromises retention of DNA repair factors

The nucleus is physically linked to the cytoskeleton, adhesions, and extracellular matrix—all of which sustain forces, but their relationships to DNA damage are obscure. We show that nuclear rupture with cytoplasmic mislocalization of multiple DNA repair factors correlates with high nuclear curvature imposed by an external probe or by cell attachment to either aligned collagen fibers or stiff matrix. Mislocalization is greatly enhanced by lamin A depletion, requires hours for nuclear reentry, and correlates with an increase in pan-nucleoplasmic foci of the DNA damage marker &ggr;H2AX. Excess DNA damage is rescued in ruptured nuclei by cooverexpression of multiple DNA repair factors as well as by soft matrix or inhibition of actomyosin tension. Increased contractility has the opposite effect, and stiff tumors with low lamin A indeed exhibit increased nuclear curvature, more frequent nuclear rupture, and excess DNA damage. Additional stresses likely play a role, but the data suggest high curvature promotes nuclear rupture, which compromises retention of DNA repair factors and favors sustained damage.

Filomicelles Deliver a Chemo-Differentiation Combination of Paclitaxel and Retinoic Acid That Durably Represses Carcinomas in Liver to Prolong Survival

Drug resistance and relapse is common in cancer treatments with chemotherapeutics, and while drug combinations with naturally occurring, differentiation-inducing retinoic acid (RA) provide remission-free cures for one type of liquid tumor, solid tumors present major problems for delivery. Here, inspired by filoviruses that can be microns in length, flexible filomicelles that self-assemble from an amphiphilic block copolymer (PEG-PCL) are shown to effectively deliver RA and paclitaxel (TAX) to several solid tumor models, particularly in the liver. These hydrophobic compounds synergistically load into the cores of the elongated micelles, and the coloaded micelles prove most effective at causing cell death, ploidy, and durable regression of tumors compared to free drugs or to separately loaded drugs. RA-TAX filomicelles also reduce mortality of human lung or liver derived cancers engrafted at liver, intraperitoneal, and subcutaneous sites in immunodeficient mice. In vitro studies show that the dual drug micelles effectively suppress proliferation while upregulating a generic differentiation marker. The results highlight the potency of dual-loaded filomicelles in killing cancer cells or else driving their differentiation away from growth.

511. Engineered Donor Marrow Macrophages Phagocytose Cancer Cells and Aggressively Shrink Solid Tumor Xenografts Compared to Tumor Associated Macrophages

Cell-based immunotherapies such as those based on engineered T-cells appear safe and often effective against liquid tumors. In solid tumors, macrophages are typically abundant, but the density of tumor associated macrophages (TAMs) correlates with poor clinical outcomes as they promote tumor growth, immunosuppression, and are nonphagocytic. In our studies, less differentiated donor marrow phagocytes are engineered to target tumors and selectively phagocytose cancer cells. Xenograft tumors were made on the flanks of NSG mice using a tdTomato human lung carcinoma cell line (A549). Systemic injections of anti-human IgG (anti-hum) with large tumors (~70 mm2) showed no effect on tumor growth. However, systemic injection of bone marrow from donor NSG mice together with biweekly anti-hum treatments effectively stopped growth of the solid tumors. Replacing anti-hum with a non-specific antibody had no effect on tumor growth. Based on tdTomato signal intensity within macrophages isolated from tumors, 10-fold more donor macrophages are phagocytic compared to resident TAMs (2-3% are phagocytic). Since cancer cells express on their surface ‘self’ markers that limit the phagocytosis of these cells, we inhibited the ‘self’ receptors on the injected donor phagocytes prior to systemic injection of the donor marrow. This combination of ‘self’-receptor inhibition with anti-hum causes a rapid decrease in tumor burden, shrinking tumors by ~40% in just 10 days compared to a similar growth of untreated tumors in the same time period. The anti-hum injection was again necessary as injection of a non-specific antibody failed to affect tumor growth. Tumor analysis showed that >85% of macrophages that were ‘self’-receptor inhibited had phagocytosed the tdTomato A549 cells, which is ~30-fold greater than resident macrophages. Importantly, these cell therapy treatments appear safe with no significant decreases in hematocrit or platelets, which is unlike the anemia that has been reported upon systemic injection of ‘self’ inhibitors. Our results thus suggest that therapies based on engineered macrophages can be safe and effective against solid tumors if three requirements are met: a phagocytic phenotype, target opsonization, and inhibition of ‘self’ signaling.