INAUGURAL ARTICLE by a Recently Elected Academy Member:The effect of parathyroid hormone on osteogenesis is mediated partly by osteolectin

We previously described a new osteogenic growth factor, osteolectin/Clec11a, which is required for the maintenance of skeletal bone mass during adulthood. Osteolectin binds to Integrin α11 (Itga11), promoting Wnt pathway activation and osteogenic differentiation by leptin receptor⁺ (LepR⁺) stromal cells in the bone marrow. Parathyroid hormone (PTH) and sclerostin inhibitor (SOSTi) are bone anabolic agents that are administered to patients with osteoporosis. Here we tested whether osteolectin mediates the effects of PTH or SOSTi on bone formation. We discovered that PTH promoted Osteolectin expression by bone marrow stromal cells within hours of administration and that PTH treatment increased serum osteolectin levels in mice and humans. Osteolectin deficiency in mice attenuated Wnt pathway activation by PTH in bone marrow stromal cells and reduced the osteogenic response to PTH in vitro and in vivo. In contrast, SOSTi did not affect serum osteolectin levels and osteolectin was not required for SOSTi-induced bone formation. Combined administration of osteolectin and PTH, but not osteolectin and SOSTi, additively increased bone volume. PTH thus promotes osteolectin expression and osteolectin mediates part of the effect of PTH on bone formation.

The maintenance and repair of the skeleton require the generation of new bone cells throughout adult life. Osteoblasts are relatively short-lived cells that are constantly regenerated, partly by skeletal stem cells within the bone marrow (1). The main source of new osteoblasts in adult bone marrow is leptin receptor-expressing (LepR⁺) stromal cells (2–4). These cells include the multipotent skeletal stem cells that give rise to the fibroblast colony-forming cells (CFU-Fs) in the bone marrow (2), as well as restricted osteogenic progenitors (5) and adipocyte progenitors (6–8). LepR⁺ cells are a major source of osteoblasts for fracture repair (2) and growth factors for hematopoietic stem cell maintenance (9–11).One growth factor synthesized by LepR⁺ cells, as well as osteoblasts and osteocytes, is osteolectin/Clec11a, a secreted glycoprotein of the C-type lectin domain superfamily (5, 12, 13). Osteolectin is an osteogenic factor that promotes the maintenance of the adult skeleton by promoting the differentiation of LepR⁺ cells into osteoblasts. Osteolectin acts by binding to integrin α11β1, which is selectively expressed by LepR⁺ cells and osteoblasts, activating the Wnt pathway (12). Deficiency for either Osteolectin or Itga11 (the gene that encodes integrin α11) reduces osteogenesis during adulthood and causes early-onset osteoporosis in mice (12, 13). Recombinant osteolectin promotes osteogenic differentiation by bone marrow stromal cells in culture and daily injection of mice with osteolectin systemically promotes bone formation.Osteoporosis is a progressive condition characterized by reduced bone mass and increased fracture risk (14). Several factors contribute to osteoporosis development, including aging, estrogen insufficiency, mechanical unloading, and prolonged glucocorticoid use (14). Existing therapies include antiresorptive agents that slow bone loss, such as bisphosphonates (15, 16) and estrogens (17), and anabolic agents that increase bone formation, such as parathyroid hormone (PTH) (18), PTH-related protein (19), and sclerostin inhibitor (SOSTi) (20). While these therapies increase bone mass and reduce fracture risk, they are not a cure.PTH promotes both anabolic and catabolic bone remodeling (21–24). PTH is synthesized by the parathyroid gland and regulates serum calcium levels, partly by regulating bone formation and bone resorption (23–25). PTH1R is a PTH receptor (26, 27) that is strongly expressed by LepR⁺ bone marrow stromal cells (8, 28–30). Recombinant human PTH (Teriparatide; amino acids 1 to 34) and synthetic PTH-related protein (Abaloparatide) are approved by the US Food and Drug Administration (FDA) for the treatment of osteoporosis (19, 31). Daily (intermittent) administration of PTH increases bone mass by promoting the differentiation of osteoblast progenitors, inhibiting osteoblast and osteocyte apoptosis, and reducing sclerostin levels (32–35). PTH promotes osteoblast differentiation by activating Wnt and BMP signaling in bone marrow stromal cells (28, 36, 37), although the mechanisms by which it regulates Wnt pathway activation are complex and uncertain (38).Sclerostin is a secreted glycoprotein that inhibits Wnt pathway activation by binding to LRP5/6, a widely expressed Wnt receptor (7, 8), reducing bone formation (39, 40). Sclerostin is secreted by osteocytes (8, 41), negatively regulating bone formation by inhibiting the differentiation of osteoblasts (41, 42). SOSTi (Romosozumab) is a humanized monoclonal antibody that binds sclerostin, preventing binding to LRP5/6 and increasing Wnt pathway activation and bone formation (43). It is FDA-approved for the treatment of osteoporosis (20, 44) and has activity in rodents in addition to humans (45, 46).The discovery that osteolectin is a bone-forming growth factor raises the question of whether it mediates the effects of PTH or SOSTi on osteogenesis.     

Characterization of the strain-rate–dependent mechanical response of single cell–cell junctions

Cell–cell adhesions are often subjected to mechanical strains of different rates and magnitudes in normal tissue function. However, the rate-dependent mechanical behavior of individual cell–cell adhesions has not been fully characterized due to the lack of proper experimental techniques and therefore remains elusive. This is particularly true under large strain conditions, which may potentially lead to cell–cell adhesion dissociation and ultimately tissue fracture. In this study, we designed and fabricated a single-cell adhesion micro tensile tester (SCAµTT) using two-photon polymerization and performed displacement-controlled tensile tests of individual pairs of adherent epithelial cells with a mature cell–cell adhesion. Straining the cytoskeleton–cell adhesion complex system reveals a passive shear-thinning viscoelastic behavior and a rate-dependent active stress-relaxation mechanism mediated by cytoskeleton growth. Under low strain rates, stress relaxation mediated by the cytoskeleton can effectively relax junctional stress buildup and prevent adhesion bond rupture. Cadherin bond dissociation also exhibits rate-dependent strengthening, in which increased strain rate results in elevated stress levels at which cadherin bonds fail. This bond dissociation becomes a synchronized catastrophic event that leads to junction fracture at high strain rates. Even at high strain rates, a single cell–cell junction displays a remarkable tensile strength to sustain a strain as much as 200% before complete junction rupture. Collectively, the platform and the biophysical understandings in this study are expected to build a foundation for the mechanistic investigation of the adaptive viscoelasticity of the cell–cell junction.

Adhesive organelles between neighboring epithelial cells form an integrated network as the foundation of complex tissues (1). As part of normal physiology, this integrated network is constantly exposed to mechanical stress and strain, which is essential to normal cellular activities, such as proliferation (2–4), migration (5, 6), differentiation (7), and gene regulation (7, 8) associated with a diverse set of functions in tissue morphogenesis (9–11) and wound healing (9). A host of developmental defects or clinical pathologies in the form of compromised cell–cell associations will arise when cells fail to withstand external mechanical stress due to genetic mutations or pathological perturbations (12, 13). Indeed, since the mechanical stresses are mainly sustained by the intercellular junctions, which may represent the weakest link and limit the stress tolerance within the cytoskeleton network of a cell sheet, mutations or disease-induced changes in junction molecules and components in adherens junctions and desmosomes lead to cell layer fracture and tissue fragility, which exacerbate the pathological conditions (14–17). This clinical relevance gives rise to the importance of understanding biophysical transformations of the cell–cell adhesion interface when cells are subjected to mechanical loads.As part of their normal functions, cells often experience strains of tens to a few hundred percent at strain rates of 10⁻⁴ to 1 s⁻¹ (18–21). For instance, embryonic epithelia are subjected to strain rates in the range of 10⁻⁴ to 10⁻³ s⁻¹ during normal embryogenesis (22). Strain rates higher than 0.1 s⁻¹ are often experienced by adult epithelia during various normal physiological functions (21, 23, 24), such as breathing motions in the lung (1 to 10 s⁻¹) (25), cardiac pulses in the heart (1 to 6.5 s⁻¹) (20), peristaltic movements in the gut (0.4 to 1.5 s⁻¹), and normal stretching of the skin (0.1 to 5 s⁻¹). Cells have different mechanisms to dissipate the internal stress produced by external strain to avoid fracture, often via cytoskeleton remodeling and cell–cell adhesion enhancement (26, 27). These coping mechanisms may have different characteristic timescales. Cytoskeleton remodeling can dissipate mechanical stress promptly due to its viscoelastic nature and the actomyosin-mediated cell contractility (17, 28–32). Adhesion enhancement at the cell–cell contact is more complex in terms of timescale. Load-induced cell–cell adhesion strengthening has been shown via the increase in the number of adhesion complexes (33–35) or by the clustering of adhesion complexes (36–39), which occurs on a timescale ranging from a few minutes up to a few hours after cells experience an initial load (28). External load on the cell–cell contact also results in a prolonged cell–cell adhesion dissociation time (40, 41), suggesting cadherin bonds may transition to catch bonds under certain loading conditions (42, 43), which can occur within seconds (44). With the increase in cellular tension, failure to dissipate the stress within the cell layer at a rate faster than the accumulation rate will inevitably lead to the fracture of the cell layer (45). Indeed, epithelial fracture often aggravates the pathological outcomes in several diseases, such as acute lung injuries (46), skin disorders (47), and development defects (48). It is generally accepted that stress accumulation in the cytoskeleton network (49, 50) and potentially in the cytoplasm is strain-rate–dependent (51). However, to date, there is a lack of understanding about the rate-dependent behavior of cell–cell adhesions, particularly about which of the stress-relaxation mechanisms are at play across the spectrum of strain rates. In addition, it remains unclear how the stress relaxation interplays with adhesion enhancement under large strains, especially at high strain rates which may lead to fracture, that is, a complete separation of mature cell–cell adhesions under a tensile load (45, 52, 53). Yet, currently, there is a lack of quantitative technology that enables the investigation of these mechanobiological processes in a precisely controlled manner. This is especially true at high strain rates.To delineate this mechanical behavior, the cleanest characterization method is to directly measure stress dynamics at a single mature cell–cell adhesion interface. Specifically, just as a monolayer cell sheet is a reduction from three-dimensional (3D) tissue, a single cell–cell adhesion interface, as a reduction from a monolayer system, represents the smallest unit to study the rheological behavior of cellular junctions. The mechanistic understanding uncovered with this single unit will inform cellular adaptations to a more complex stress microenvironment in vivo and in vitro, in healthy and diseased conditions. To this end, we developed a single-cell adhesion micro tensile tester (SCAµTT) platform based on nanofabricated polymeric structures using two-photon polymerization (TPP). This platform allows in situ investigation of stress–strain characteristics of a mature cell–cell junction through defined strains and strain rates. With SCAµTT, we reveal some interesting biophysical phenomena at the single cell–cell junction that were previously not possible to observe using existing techniques. We show that cytoskeleton growth can effectively relax intercellular stress between an adherent cell pair in a strain-rate–dependent manner. Along with cadherin-clustering–induced bond strengthening, it prevents failure to occur at low strain rates. At high strain rates, insufficient relaxation leads to stress accumulation, which results in cell–cell junction rupture. We show that a remarkably large strain can be sustained before junction rupture (>200%), even at a strain rate as high as 0.5 s⁻¹. Collectively, the rate-dependent mechanical characterization of the cell–cell junction builds the foundation for an improved mechanistic understanding of junction adaptation to an external load and potentially the spatiotemporal coordination of participating molecules at the cell–cell junction.     

SLFN11 promotes CDT1 degradation by CUL4 in response to replicative DNA damage,while its absence leads to synthetic lethality with ATR/CHK1 inhibitors

Schlafen-11 (SLFN11) inactivation in ∼50% of cancer cells confers broad chemoresistance. To identify therapeutic targets and underlying molecular mechanisms for overcoming chemoresistance, we performed an unbiased genome-wide RNAi screen in SLFN11-WT and -knockout (KO) cells. We found that inactivation of Ataxia Telangiectasia- and Rad3-related (ATR), CHK1, BRCA2, and RPA1 overcome chemoresistance to camptothecin (CPT) in SLFN11-KO cells. Accordingly, we validate that clinical inhibitors of ATR (M4344 and M6620) and CHK1 (SRA737) resensitize SLFN11-KO cells to topotecan, indotecan, etoposide, cisplatin, and talazoparib. We uncover that ATR inhibition significantly increases mitotic defects along with increased CDT1 phosphorylation, which destabilizes kinetochore-microtubule attachments in SLFN11-KO cells. We also reveal a chemoresistance mechanism by which CDT1 degradation is retarded, eventually inducing replication reactivation under DNA damage in SLFN11-KO cells. In contrast, in SLFN11-expressing cells, SLFN11 promotes the degradation of CDT1 in response to CPT by binding to DDB1 of CUL4^CDT2 E3 ubiquitin ligase associated with replication forks. We show that the C terminus and ATPase domain of SLFN11 are required for DDB1 binding and CDT1 degradation. Furthermore, we identify a therapy-relevant ATPase mutant (E669K) of the SLFN11 gene in human TCGA and show that the mutant contributes to chemoresistance and retarded CDT1 degradation. Taken together, our study reveals new chemotherapeutic insights on how targeting the ATR pathway overcomes chemoresistance of SLFN11-deficient cancers. It also demonstrates that SLFN11 irreversibly arrests replication by degrading CDT1 through the DDB1–CUL4^CDT2 ubiquitin ligase.

Schlafen-11 (SLFN11) is an emergent restriction factor against genomic instability acting by eliminating cells with replicative damage (1–6) and potentially acting as a tumor suppressor (6, 7). SLFN11-expressing cancer cells are consistently hypersensitive to a broad range of chemotherapeutic drugs targeting DNA replication, including topoisomerase inhibitors, alkylating agents, DNA synthesis, and poly(ADP-ribose) polymerase (PARP) inhibitors compared to SLFN11-deficient cancer cells, which are chemoresistant (1, 2, 4, 8–17). Profiling SLFN11 expression is being explored for patients to predict survival and guide therapeutic choice (8, 13, 18–24).The Cancer Genome Atlas (TCGA) and cancer cell databases demonstrate that SLFN11 mRNA expression is suppressed in a broad fraction of common cancer tissues and in ∼50% of all established cancer cell lines across multiple histologies (1, 2, 5, 8, 13, 25, 26). Silencing of the SLFN11 gene, like known tumor suppressor genes, is under epigenetic mechanisms through hypermethylation of its promoter region and activation of histone deacetylases (HDACs) (21, 23, 25, 26). A recent study in small-cell lung cancer patient-derived xenograft models also showed that SLFN11 gene silencing is caused by local chromatin condensation related to deposition of H3K27me3 in the gene body of SLFN11 by EZH2, a histone methyltransferase (11). Targeting epigenetic regulators is therefore an attractive combination strategy to overcome chemoresistance of SLFN11-deficient cancers (10, 25, 26). An alternative approach is to attack SLFN11-negative cancer cells by targeting the essential pathways that cells use to overcome replicative damage and replication stress. Along these lines, a prior study showed that inhibition of ATR (Ataxia Telangiectasia- and Rad3-related) kinase reverses the resistance of SLFN11-deficient cancer cells to PARP inhibitors (4). However, targeting the ATR pathway in SLFN11-deficient cells has not yet been fully explored.SLFN11 consists of two functional domains: A conserved nuclease motif in its N terminus and an ATPase motif (putative helicase) in its C terminus (2, 6). The N terminus nuclease has been implicated in the selective degradation of type II tRNAs (including those coding for ATR) and its nuclease structure can be derived from crystallographic analysis of SLFN13 whose N terminus domain is conserved with SLFN11 (27, 28). The C terminus is only present in the group III Schlafen family (24, 29). Its potential ATPase activity and relationship to chemosensitivity to DNA-damaging agents (3–5) imply that the ATPase/helicase of SLFN11 is involved specifically in DNA damage response (DDR) to replication stress. Indeed, inactivation of the Walker B motif of SLFN11 by the mutation E669Q suppresses SLFN11-mediated replication block (5, 30). In addition, SLFN11 contains a binding site for the single-stranded DNA binding protein RPA1 (replication protein A1) at its C terminus (3, 31) and is recruited to replication damage sites by RPA (3, 5). The putative ATPase activity of SLFN11 is not required for this recruitment (5) but is required for blocking the replication helicase complex (CMG-CDC45) and inducing chromatin accessibility at replication origins and promoter sites (5, 30). Based on these studies, our current model is that SLFN11 is recruited to “stressed” replication forks by RPA filaments formed on single-stranded DNA (ssDNA), and that the ATPase/helicase activity of SLFN11 is required for blocking replication progression and remodeling chromatin (5, 30). However, underlying mechanisms of how SLFN11 irreversibly blocks replication in DNA damage are still unclear.Increased RPA-coated ssDNA caused by DNA damage and replication fork stalling also triggers ATR kinase activation, promoting subsequent phosphorylation of CHK1, which transiently halts cell cycle progression and enables DNA repair (32). ATR inhibitors are currently in clinical development in combination with DNA replication damaging drugs (33, 34), such as topoisomerase I (TOP1) inhibitors, which are highly synergistic with ATR inhibitors in preclinical models (35). ATR inhibitors not only inhibit DNA repair, but also lead to unscheduled replication origin firing (36), which kills cancer cells (37, 38) by inducing genomic alterations due to faulty replication and mitotic catastrophe (33).The replication licensing factor CDT1 orchestrates the initiation of replication by assembling prereplication complexes (pre-RC) in G1-phase before cells enter S-phase (39). Once replication is started by loading and activation of the MCM helicase, CDT1 is degraded by the ubiquitin proteasomal pathway to prevent additional replication initiation and ensure precise genome duplication and the firing of each origin only once per cell cycle (39, 40). At the end of G2 and during mitosis, CDT1 levels rise again to control kinetochore-microtubule attachment for accurate chromosome segregation (41). Deregulated overexpression of CDT1 results in rereplication, genome instability, and tumorigenesis (42). The cellular CDT1 levels are tightly regulated by the damage-specific DNA binding protein 1 (DDB1)–CUL4^CDT2 E3 ubiquitin ligase complex in G1-phase (43) and in response to DNA damage (44, 45). How CDT1 is recognized by CUL4^CDT2 in response to DNA damage remains incompletely known.In the present study, starting with a human genome-wide RNAi screen, bioinformatics analyses, and mechanistic validations, we explored synthetic lethal interactions that overcome the chemoresistance of SLFN11-deficient cells to the TOP1 inhibitor camptothecin (CPT). The strongest synergistic interaction was between depletion of the ATR/CHK1-mediated DNA damage response pathways and DNA-damaging agents in SLFN11-deficient cells. We validated and expanded our molecular understanding of combinatorial strategies in SLFN11-deficient cells with the ATR (M4344 and M6620) and CHK1 (SRA737) inhibitors in clinical development (33, 46, 47) and found that ATR inhibition leads to CDT1 stabilization and hyperphosphorylation with mitotic catastrophe. Our study also establishes that SLFN11 promotes the degradation of CDT1 by binding to DDB1, an adaptor molecule of the CUL4^CDT2 E3 ubiquitin ligase complex, leading to an irreversible replication block in response to replicative DNA damage.     

Collagen IV differentially regulates planarian stem cell potency and lineage progression

The extracellular matrix (ECM) provides a precise physical and molecular environment for cell maintenance, self-renewal, and differentiation in the stem cell niche. However, the nature and organization of the ECM niche is not well understood. The adult freshwater planarian Schmidtea mediterranea maintains a large population of multipotent stem cells (neoblasts), presenting an ideal model to study the role of the ECM niche in stem cell regulation. Here we tested the function of 165 planarian homologs of ECM and ECM-related genes in neoblast regulation. We identified the collagen gene family as one with differential effects in promoting or suppressing proliferation of neoblasts. col4-1, encoding a type IV collagen α-chain, had the strongest effect. RNA interference (RNAi) of col4-1 impaired tissue maintenance and regeneration, causing tissue regression. Finally, we provide evidence for an interaction between type IV collagen, the discoidin domain receptor, and neuregulin-7 (NRG-7), which constitutes a mechanism to regulate the balance of symmetric and asymmetric division of neoblasts via the NRG-7/EGFR pathway.

Across the animal kingdom, stem cell function is regulated by the microenvironment in the surrounding niche (1), where the concentration of molecular signals for self-renewal and differentiation can be precisely regulated (2). The niche affects stem cell biology in many processes, such as aging and tissue regeneration, as well as pathological conditions such as cancer (3). Most studies have been done in tissues with large stem cell populations, such as the intestinal crypt (4) and the hair follicle (5) in mice. Elucidation of the role of the stem cell niche in tissue regeneration requires the study of animals with high regenerative potential, such as freshwater planarians (flatworms) (6). Dugesia japonica and Schmidtea mediterranea are two well-studied species that possess the ability to regenerate any missing body part (6, 7).Adult S. mediterranea maintain a high number of stem cells (neoblasts)—∼10 to 30% of all somatic cells in the adult worm—with varying potency, including pluripotent cells (8–14). Neoblasts are the only proliferating somatic cells: they are molecularly heterogeneous, but all express piwi-1 (15–18). Lineage-committed neoblasts are “progenitors” that transiently express both piwi-1 and tissue-specific genes (15, 19). Examples include early intestinal progenitors (γ neoblast, piwi-1⁺/hnf4⁺) (8, 10, 15, 19–21) and early epidermal progenitors (ζ neoblast, piwi-1⁺/zfp-1⁺) (8, 15). Other progenitor markers include collagen for muscles (22), ChAT for neurons (23), and cavII for protonephridia (24, 25). During tissue regeneration, neoblasts are recruited to the wound site, where they proliferate then differentiate to replace the missing cell types (16, 26). Some neoblasts express the pluripotency marker tgs-1, and are designated as clonogenic neoblasts (cNeoblasts) (10, 11). cNeoblasts are located in the parenchymal space adjacent to the gut (11).Neoblasts are sensitive to γ-irradiation and can be preferentially depleted in the adult planarian (27). After sublethal γ-irradiation, remaining cNeoblasts can repopulate the stem cell pool within their niche (10, 11). The close proximity of neoblasts to the gut suggests gut may be a part of neoblast niche (28, 29). When gut integrity was impaired by silencing gata4/5/6, the egfr-1/nrg-1 ligand-receptor pair, or wwp1, maintenance of non–γ-neoblasts were also disrupted (20, 30, 31), but whether that indicates the gut directly regulates neoblast remains unclear. There is evidence indicating the dorsal-ventral (D/V) transverse muscles surrounding the gut may promote neoblast proliferation and migration, with the involvement of matrix metalloproteinase mt-mmpB (32, 33). The central nervous system has also been implicated in influencing neoblast maintenance through the expression of EGF homolog neuregulin-7 (nrg-7), a ligand for EGFR-3, affecting the balance of neoblast self-renewal (symmetric or asymmetric division) (34).In other model systems, an important component of the stem-cell niche is the extracellular matrix (ECM) (35). Germline stem cells in Drosophila are anchored to niche supporting cells with ECM on one side, while the opposite side is exposed to differentiation signals, allowing asymmetric cell fate outcomes for self-renewal or differentiation following division (36–38). Few studies have addressed the ECM in planarians, largely due to the lack of genetic tools to manipulate the genome, the absence of antibodies to specific planarian ECM homologs, or the tools required to study cell fate changes. However, the genomes of D. japonica (39–41) and S. mediterranea (41–45), and single-cell RNA-sequencing (scRNA-seq) datasets for S. mediterranea are now available (11, 46–50). A recent study of the planarian matrisome demonstrated that muscle cells are the primary source of many ECM proteins (51), which, together with those produced by neoblasts and supporting parenchymal cells, may constitute components of the neoblast niche. For example, megf6 and hemicentin restrict neoblast’s localization within the parenchyma (51, 52). Functional studies also implicate ECM-modifiers, such as matrix metalloproteases (MMPs) in neoblast migration and regeneration. For example, reducing the activity of the ECM-degrading enzymes mt-mmpA (26, 33), mt-mmpB (53), or mmp-1 (33) impaired neoblast migration, proliferation, or overall tissue growth, respectively. Neoblasts are also likely to interact with ECM components of the niche via cell surface receptors, such as β1 integrin, inactivation of which impairs brain regeneration (54, 55).Here, we identified planarian ECM homologs in silico, followed by systematic functional assessment of 165 ECM and ECM-related genes by RNA interference (RNAi), to determine the effect on neoblast repopulation in planarians challenged by a sublethal dose of γ-irradiation (10). Surprisingly, multiple classes of collagens were shown to have the strongest effects. In particular, we show that the type IV collagens (COLIV) of basement membranes (BMs), were required to regulate the repopulation of neoblasts as well as lineage progression to progenitor cells. Furthermore, our data support an interaction between COLIV and the discoidin domain receptor (DDR) in neurons that activates signaling of NRG-7 in the neoblasts to regulate neoblast self-renewal versus differentiation. Together, these data demonstrate multifaceted regulation of planarian stem cells by ECM components.     

From the Cover: Radiation-induced neoantigens broaden the immunotherapeutic window of cancers with low mutational loads

Immunotherapies are a promising advance in cancer treatment. However, because only a subset of cancer patients benefits from these treatments it is important to find mechanisms that will broaden the responding patient population. Generally, tumors with high mutational burdens have the potential to express greater numbers of mutant neoantigens. As neoantigens can be targets of protective adaptive immunity, highly mutated tumors are more responsive to immunotherapy. Given that external beam radiation 1) is a standard-of-care cancer therapy, 2) induces expression of mutant proteins and potentially mutant neoantigens in treated cells, and 3) has been shown to synergize clinically with immune checkpoint therapy (ICT), we hypothesized that at least one mechanism of this synergy was the generation of de novo mutant neoantigen targets in irradiated cells. Herein, we use Kras^G12D x p53^−/− sarcoma cell lines (KP sarcomas) that we and others have shown to be nearly devoid of mutations, are poorly antigenic, are not controlled by ICT, and do not induce a protective antitumor memory response. However, following one in vitro dose of 4- or 9-Gy irradiation, KP sarcoma cells acquire mutational neoantigens and become sensitive to ICT in vivo in a T cell-dependent manner. We further demonstrate that some of the radiation-induced mutations generate cytotoxic CD8⁺ T cell responses, are protective in a vaccine model, and are sufficient to make the parental KP sarcoma line susceptible to ICT. These results provide a proof of concept that induction of new antigenic targets in irradiated tumor cells represents an additional mechanism explaining the clinical findings of the synergy between radiation and immunotherapy.

Immune checkpoint therapy (ICT) can lead to durable responses in subsets of cancer patients (1–8). On the basis of computational analyses, the patients who most benefit from ICT are those with cancers that have high mutational burden (9–18). For example, patients bearing tumors with high mutational burden caused by environmental exposure (such as ultraviolet-induced melanoma) or deficiencies in DNA repair (such as microsatellite instability-high colorectal cancers) tend to respond well to immunotherapy (18–26). Presumably the sensitivity of such cancers reflects the increased likelihood of formation of immunogenic, tumor-specific mutant neoantigens (27). We and others previously showed that certain tumor-specific neoantigens are major targets of natural and therapeutically induced antitumor responses in both mice and humans (28–41). Therefore, the presence of immunogenic tumor neoantigens is currently thought to contribute to tumor sensitivity to immunotherapy.However, many cancer patients do not respond to ICT, suggesting that their neoantigen burden is either of insufficient magnitude or immunogenicity to function as targets for T cell-dependent antitumor mechanisms. Indeed, there are many tumor types, such as acute myeloid leukemia, estrogen receptor-positive breast, and prostate cancers, that have limited mutational burdens and display low response rates to ICT (9, 13, 42, 43). Additionally, tumor cell clones expressing immunogenic neoantigens that develop during tumor evolution may be eliminated from tumors with high mutational burden by the process of cancer immunoediting, resulting in outgrowth of tumor cell clones with reduced immunogenicity that can then grow progressively in the presence of the unmanipulated immune system (33, 44, 45). Therefore, a process by which tumors with low neoantigen burden can acquire immunogenic mutations has the potential to expand the number of patients able to benefit from ICT.Ionizing radiation has been shown to elicit DNA damage in tumor cells, leading to an increase in overall mutational load (46–52). This damage is thought to occur primarily through generation of reactive oxygen species which induce base pair substitutions by mechanisms involving transitions, transversions, and/or faulty DNA repair (53). Multiple preclinical studies have demonstrated antitumor responses when focal radiation is combined with ICT in tumors that do not respond to ICT alone (54–60) and several clinical studies have demonstrated that human tumor patients have improved responsiveness to ICT following focal radiation (e.g., {"type":"clinical-trial","attrs":{"text":"NCT02303990","term_id":"NCT02303990"}}NCT02303990, {"type":"clinical-trial","attrs":{"text":"NCT02298946","term_id":"NCT02298946"}}NCT02298946, {"type":"clinical-trial","attrs":{"text":"NCT02383212","term_id":"NCT02383212"}}NCT02383212) (61–67). Radiation has been demonstrated to function as an in vivo tumor vaccine by inducing damage-associated molecular patterns (DAMP)-dependent immunogenic cell death (68), inducing DNA damage sensed by pattern recognition receptors (69, 70), enhancing access of immune effector cells to their cognate targets through tumor cell debulking and vasculature changes (71, 72), up-regulating major histocompatibility complex class I (MHC-I) receptors (73), up-regulating cell-surface molecules such as Fas (74), and augmenting tumor antigen cross-presentation by specific subsets of dendritic cells through up-regulation of type I interferon (IFN), which results in increased numbers and action of tumor-specific CD8⁺ T cells (75–77). However, none of these explanations take into account that following irradiation, tumor cells acquire novel mutations that may function as effective tumor neoantigens. In fact, two groups have demonstrated broadening of the T cell repertoire following radiation treatment of mouse 4T1 mammary tumors and B16F10 melanoma tumors (56, 78). Radiation-induced neoantigens may partially explain the broadening of the T cell repertoire reported during noncurative doses of irradiation.Given the above observations, we specifically explored whether one dose of in vitro irradiation could increase the immunogenicity of poorly immunogenic tumor cell lines through mechanisms involving the de novo generation of tumor-specific mutant neoantigens. For this purpose, we used a mouse Kras^G12D x p53^−/− sarcoma cell line as a model system since the R.D.S. and T.J. laboratories have previously shown that these tumor cells express a very limited number of somatic mutations, are essentially devoid of mutational neoantigens, and are nonimmunogenic and grow progressively in syngeneic wild-type (WT) mice either following treatment with control antibody or the combination of anti–PD-1/anti–CTLA-4 (34, 41). We find that treating these cell lines with noncurative doses of irradiation induces expression of somatic mutations, some of which function as neoantigens and render the sarcoma cells susceptible to ICT in vivo. These data support the concept that an additional mechanism underlying the synergy between radiation therapy and immunotherapy is that the former induces immunogenic mutations in tumors that now function as targets for the latter.     

Violet light suppresses lens-induced myopia via neuropsin (OPN5) in mice

Myopia has become a major public health concern, particularly across much of Asia. It has been shown in multiple studies that outdoor activity has a protective effect on myopia. Recent reports have shown that short-wavelength visible violet light is the component of sunlight that appears to play an important role in preventing myopia progression in mice, chicks, and humans. The mechanism underlying this effect has not been understood. Here, we show that violet light prevents lens defocus–induced myopia in mice. This violet light effect was dependent on both time of day and retinal expression of the violet light sensitive atypical opsin, neuropsin (OPN5). These findings identify Opn5-expressing retinal ganglion cells as crucial for emmetropization in mice and suggest a strategy for myopia prevention in humans.

Myopia (nearsightedness) in school-age children is generally axial myopia, which is the consequence of elongation of the eyeball along the visual axis. This shape change results in blurred vision but can also lead to severe complications including cataract, retinal detachment, myopic choroidal neovascularization, glaucoma, and even blindness (1–3). Despite the current worldwide pandemic of myopia, the mechanism of myopia onset is still not understood (4–8). One hypothesis that has earned a current consensus is the suggestion that a change in the lighting environment of modern society is the cause of myopia (9, 10). Consistent with this, outdoor activity has a protective effect on myopia development (9, 11, 12), though the main reason for this effect is still under debate (7, 12, 13). One explanation is that bright outdoor light can promote the synthesis and release of dopamine in the eye, a myopia-protective neuromodulator (14–16). Another suggestion is that the distinct wavelength composition of sunlight compared with fluorescent or LED (light-emitting diode) artificial lighting may influence myopia progression (9, 10). Animal studies have shown that different wavelengths of light can affect the development of myopia independent of intensity (17, 18). The effects appear to be distinct in different species: for chicks and guinea pigs, blue light showed a protective effect on experimentally induced myopia, while red light had the opposite effect (18–22). For tree shrews and rhesus monkeys, red light is protective, and blue light causes dysregulation of eye growth (23–25).It has been shown that visible violet light (VL) has a protective effect on myopia development in mice, in chick, and in human (10, 26, 27). According to Commission Internationale de l’Eclairage (International Commission on Illumination), VL has the shortest wavelength of visible light (360 to 400 nm). These wavelengths are abundant in outside sunlight but can only rarely be detected inside buildings. This is because the ultraviolet (UV)-protective coating on windows blocks all light below 400 nm and because almost no VL is emitted by artificial light sources (10). Thus, we hypothesized that the lack of VL in modern society is one reason for the myopia boom (9, 10, 26).In this study, we combine a newly developed lens-induced myopia (LIM) model with genetic manipulations to investigate myopia pathways in mice (28, 29). Our data confirm (10, 26) that visible VL is protective but further show that delivery of VL only in the evening is sufficient for the protective effect. In addition, we show that the protective effect of VL on myopia induction requires OPN5 (neuropsin) within the retina. The absence of retinal Opn5 prevents lens-induced, VL-dependent thickening of the choroid, a response thought to play a key role in adjusting the size of the eyeball in both human and animal myopia models (30–33). This report thus identifies a cell type, the Opn5 retinal ganglion cell (RGC), as playing a key role in emmetropization. The requirement for OPN5 also explains why VL has a protective effect on myopia development.     

Label-free adaptive optics imaging of human retinal macrophage distribution and dynamics

Microglia are resident central nervous system macrophages and the first responders to neural injury. Until recently, microglia have been studied only in animal models with exogenous or transgenic labeling. While these studies provided a wealth of information on the delicate balance between neuroprotection and neurotoxicity within which these cells operate, extrapolation to human immune function has remained an open question. Here we examine key characteristics of retinal macrophage cells in live human eyes, both healthy and diseased, with the unique capabilities of our adaptive optics–optical coherence tomography approach and owing to their propitious location above the inner limiting membrane (ILM), allowing direct visualization of cells. Our findings indicate that human ILM macrophage cells may be distributed distinctly, age differently, and have different dynamic characteristics than microglia in other animals. For example, we observed a macular pattern that was sparse centrally and peaked peripherally in healthy human eyes. Moreover, human ILM macrophage density decreased with age (∼2% of cells per year). Our results in glaucomatous eyes also indicate that ILM macrophage cells appear to play an early and regionally specific role of nerve fiber layer phagocytosis in areas of active disease. While we investigate ILM macrophage cells distinct from the larger sample of overall retinal microglia, the ability to visualize macrophage cells without fluorescent labeling in the live human eye represents an important advance for both ophthalmology and neuroscience, which may lead to novel disease biomarkers and new avenues of exploration in disease progression.

Glial cells are nonneuronal support cells in the central nervous system (CNS) (1, 2) that include macroglia (astrocytes, ependymal cells, oligodendrocytes, and radial cells) and microglia. These cells are derived during development from different sources: macroglia from the neuroectoderm and microglia from primitive yolk sac progenitors (3, 4). In the retina, glial cell types include Müller cells, a radial cell that spans the length of the entire retina (5, 6); astrocytes, found primarily in the nerve fiber layer (NFL) (7); and microglia, a type of macrophage that reside primarily in the inner plexiform layer (IPL) and outer plexiform layer (OPL), but also in a smaller proportion in the ganglion cell layer (GCL), in the NFL, and near the inner limiting membrane (ILM) (8). Vitreous macrophages, known as hyalocytes (9), exist in two distinct morphological (and functional) subgroups, depending on their location in the vitreous and anteriorly near the ciliary body (ovoidal cells with microvilli and short cytoplasmic protrusions) or posteriorly at the ILM (larger, elongated cells with thick processes) (10, 11). When hyalocytes are imaged together with retinal microglia, they have been described as cells without ramification (12). Thus, reports on vitreous macrophage characteristics vary, probably depending on the subgroup studied. Regardless of classification, there is strong evidence for their role in epiretinal membrane formation (13, 14).While retinal microglia and ILM macrophages are typically defined according to the side of the ILM on which they reside (i.e., within or outside the CNS), they share similar features in terms of morphology, capability for migration, and process motility (15, 16). There is also strong evidence that labeled retinal microglia exist above the ILM in both mice and nonhuman primates (NHPs) (12, 17) and may be the same cells as ILM macrophages reported in the literature. In this paper, we use the terms “vitreous hyalocytes,” “ILM macrophage cells,” and “retinal microglia” to distinguish among free hyalocytes in the vitreous (and those on the epithelial surface of the ciliary body), macrophages that exist at the ILM surface, and microglia embedded within the retinal parenchyma. This nomenclature may require updating after further immunohistochemistry studies provide definitive classification of ILM macrophages as hyalocytes, retinal microglia, or a unique macrophage cell type. Furthermore, because ILM macrophage cells have such close morphological and functional correspondence to true retinal microglia embedded within the neuropil, we compare their characteristics with those of retinal microglia where appropriate throughout the paper.In the retina, as in the rest of the CNS, microglia are the primary resident immune cells and play key roles in homeostasis, neuroprotection, and neuronal cell death (8, 18, 19). Microglia assume a variety of conformational shapes to fulfill these functions and are broadly active in two different phenotypes, depending on whether they serve a neuroprotective or a neurotoxic role (20). In the neuroprotective phenotype, microglia are ramified cells whose morphology takes on a dendritic appearance with processes that constantly probe the local environment, releasing anti-inflammatory and neurotrophic factors (21, 22). In contrast, in the neurotoxic phenotype, microglia retract their processes and take on an ameboid shape, migrating to the site of injury, infection, or disease where they release inflammatory factors and become highly phagocytotic. Activated, reprogrammed microglia that migrate to the retinal pigment epithelium also act in a cytoprotective fashion that indicates their complex, multifaceted nature (23). Microglia play roles in neuroplasticity (24), cell phagocytosis (25, 26), neuroinflammatory processes (27), antigen presentation (28), complement system activation (29), angiogenesis (30), and retinal homeostasis (31). Several recent studies have also indicated that retinal microglia have regional specialization (17, 23).Microglia are involved in neurodegenerative disease processes (8, 19, 20) and, like other long-lived cells, change with senescence (18). In age-related diseases like Alzheimer’s disease, Parkinson’s disease, glaucoma, and age-related macular degeneration, the effects are interrelated (19, 20). Microglia are involved in the pathogenesis and progression of several retinal diseases (8, 20). In age-related macular degeneration, IPL and OPL microglia migrate to the subretinal space and participate in photoreceptor degeneration and drusen formation (8, 32, 33) or neovascularization (34), with subsequent inner retina replenishment from recruited monocytes (35). Elevation of proinflammatory cytokines in diabetic retinopathy is associated with microglia activation and aggregation in areas most affected during disease progression (36, 37). Microglia activation has a large role in pathogenesis of retinopathy of prematurity, in both hypoxia and hyperoxygenation (8, 38). In inherited retinal disorders such as retinitis pigmentosa, following rod photoreceptor apoptosis, microglia migrate from the inner to the outer retina, participate in rod phagocytosis, and may be implicated in subsequent cone photoreceptor cell death or phagoptosis (39). Microglia are also implicated in the pathogenesis of the family of axonopathies known as glaucoma, which result in retinal ganglion cell (RGC) death (3, 40). While glaucoma has historically been considered a disease caused primarily by the mechanical effects of elevated intraocular pressure or alternatively dysregulation of ocular blood flow (41), there are several lines of evidence indicating that neuroinflammation, including microgliosis, is closely involved in its pathogenesis (42, 43). Because microglia play such a pivotal role in disease progression, many studies have suggested that they are a prime target for neuroprotective strategies (1, 8, 20, 44).Changes in microglia occur both with age-related neurological and retinal diseases like Alzheimer’s disease, Parkinson’s disease, glaucoma, and age-related macular degeneration, but also with natural aging processes (19, 20, 25). Studies in both mice and NHPs have shown that retinal microglia density increases with age (17, 34). Other microglial structural and dynamic changes with aging include reduced branching, shorter processes, and slower migration and motility, leading to an overall deficit of their surveillance function (45). This leads to a dysfunction in microglia injury response capabilities, also manifest by aggregation, rather than dispersion, at injury sites following activation (45). The disruption in the normal activation response of microglia may involve down-regulation of proinflammatory cytokines and other neuroprotective genes (46).Until very recently, microglia required labeling for direct visualization, and so they have been studied predominantly in the mouse despite the essential roles they play in human disease progression and aging. Liu et al. (47) were the first to resolve macrophages at the ILM in the live human eye with the aid of adaptive optics–optical coherence tomography (AO-OCT). A more recent AO-OCT study has begun to explore their longer-term migration (48). Castanos et al. (16) imaged these same ILM macrophage cells in healthy controls and retinopathy patients using clinical OCT. In these early reports of human ILM macrophage imaging, extensive characterization of distribution, motility, and changes with aging and pathology were not examined.Here we begin to address this gap using the powerful combination of AO-OCT imaging and our analysis approach. Adaptive optics provides the ocular aberration correction, high transverse resolution, and precise focusing necessary to resolve ILM macrophage soma, while OCT provides the optical depth sectioning needed to visualize cells and processes in their precise axial location above the ILM. Our analysis approach includes subcellular accuracy volumetric registration using the spatial contrast collectively provided by multiple retinal layers. Indeed, a fully registered RGC mosaic may have been a prerequisite to ILM macrophage visualization, owing to its proximity to the ILM and as a cellular target in the inner retina for precise registration. High-speed acquisition and optimal temporal sampling and averaging further enhance process contrast for motility quantification. Imaging live human ILM macrophage cells without fluorescent markers was also aided by their favorable position and optical properties relative to surrounding tissue. ILM macrophage cells are not embedded within the neural parenchyma as they are in all other CNS locations in which they reside.The aim of this study was to exploit this new capability to begin to reexamine some of the characteristics of macrophage cells that have been gathered from decades of ex vivo and in vivo investigations. In particular, we sought to characterize the distribution of ILM macrophage cells across the macula, the effect of aging on distribution, the fast and slow dynamics of their motility, and their response to ocular disease. The ability to visualize and track these important immunocompetent cells in a live human eye represents an important advance in our ability to explore infection, disease, and aging processes.