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Purpose:PD-1 inhibition results in durable antitumor responses in a proportion of patients with metastatic urothelial cancer (mUC). The majority of patients, however, do not experience clinical benefit. In this study, we aimed to identify early changes in T-cell subsets that underlie anti–PD-1 efficacy in patients with mUC.Experimental Design:Paired samples were collected from peripheral blood, plasma, and metastatic lesions of 56 patients with mUC at baseline and weeks 6 and 12 after initiating pembrolizumab treatment (200 mg intravenously, every 3 weeks). Samples were analyzed using multiplex flow cytometry, ELISA, and in situ stainings, including cellular network analysis. Treatment response was evaluated as best overall response according to RECIST v1.1, and patients were classified as responder (complete or partial response) or nonresponder (progressive disease).Results:In responders, baseline fractions of CD4+ T cells expressing cosignaling receptors were higher compared with nonresponders. The fraction of circulating PD-1+ CD4+ T cells decreased at weeks 6 and 12, whereas the fraction of 4-1BB+ CD28+ CD4+ T cells increased at week 12. In metastatic lesions of responders, the baseline density of T helper-type 1 (Th1) cells, defined as T-bet+ CD4+ T cells, was higher as compared to non-responders. Upon treatment, Th1 cells became localized in close proximity to CD8+ T cells, CD11b+ myeloid cells, and tumor cells.Conclusions:A decrease in the fraction of circulating PD-1+ CD4+ T cells, and juxtaposition of Th1, CD8+, and myeloid cells was associated with response to anti–PD-1 treatment in patients with mUC.

Clusters of peripheral blood CD4+ T-cell subsets in mUC patients with and without a tumor response to anti-PD1

Numbers of major T-cell subsets in peripheral blood and expression of T-cell maturation/activation markers on CD4+ T-cells

Study design

Highlighted immune parameters in blood, plasma and tumor tissue from mUC patients with stable disease as best overall response to anti-PD1

Transcriptomic analysis of immune cell fractions, T-cell subsets, and chemo-attractants of baseline metastatic tumor biopsies from mUC patients

Immune parameters in blood, plasma and tumor tissue from mUC patients with stable disease as best overall response to anti-PD1

Proposed mechanism of action of pembrolizumab in mUC patients

Correlations between immune parameters in blood, plasma and tumor tissue

Legends to supplementary figure 1 - 9

CD20+ B-cells are present in immune cell niches in lesions of responders upon anti-PD1 treatment

Immune cells were mainly present in stroma areas of metastatic lesions of mUC patients.

Radiotherapy (RT) is the standard-of-care for Epstein–Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC), where the post-RT clearance of plasma EBV DNA is prognostic. Currently, it is not known whether the post-RT clearance of plasma EBV DNA is related to the presence of circulating T-cell subsets. Blood samples from NPC patients were used to assess the frequency of T-cell subsets relating to differentiation, co-signaling and chemotaxis. Patients with undetectable versus detectable plasma EBV DNA levels post-RT were categorized as clearers vs. non-clearers. Clearers had a lower frequency of PD1+CD8+ T cells as well as CXCR3+CD8+ T cells during RT compared to non-clearers. Clearers exclusively showed a temporal increase in chemo-attractant receptors CCR1, 4 and/or 5, expressing CD8+ T cells upon RT. The increase in CCR-expressing CD8+ T cells was accompanied by a drop in naïve CD8+ T cells and an increase in OX40+CD8+ T cells. Upon stratifying these patients based on clinical outcome, the dynamics of CCR-expressing CD8+ T cells were in concordance with the non-recurrence of NPC. In a second cohort, non-recurrence associated with higher quantities of circulating CCL14 and CCL15. Collectively, our findings relate plasma EBV DNA clearance post-RT to T-cell chemotaxis, which requires validation in larger cohorts.

Background Glioblastoma (GBM) is the most aggressive primary brain tumor. Its cellular composition is very heterogeneous, with cells exhibiting stem-cell characteristics (GSCs) that co-determine therapy resistance and tumor recurrence. Bone Morphogenetic Protein (BMP)-4 promotes astroglial and suppresses oligodendrocyte differentiation in GSCs, processes associated with superior patient prognosis. We characterized variability in cell viability of patient-derived GBM cultures in response to BMP4 and, based on single-cell transcriptome profiling, propose predictive positive and early-response markers for sensitivity to BMP4. Methods Cell viability was assessed in 17 BMP4-treated patient-derived GBM cultures. In two cultures, one highly-sensitive to BMP4 (high therapeutic efficacy) and one with low-sensitivity, response to treatment with BMP4 was characterized. We applied single-cell RNA-sequencing, analyzed the relative abundance of cell clusters, searched for and identified the aforementioned two marker types, and validated these results in all 17 cultures. Results High variation in cell viability was observed after treatment with BMP4. In three cultures with highest sensitivity for BMP4, a substantial new cell subpopulation formed. These cells displayed decreased cell proliferation and increased apoptosis. Neuronal differentiation was reduced most in cultures with little sensitivity for BMP4. OLIG1/2 levels were found predictive for high sensitivity to BMP4. Activation of ribosomal translation (RPL27A, RPS27) was up-regulated within one day in cultures that were very sensitive to BMP4. Conclusion The changes in composition of patient-derived GBM cultures obtained after treatment with BMP4 correlate with treatment efficacy. OLIG1/2 expression can predict this efficacy, and upregulation of RPL27A and RPS27 are useful early-response markers.

PD1 inhibition is effective in patients with metastatic urothelial cancer (mUC), yet a large fraction of patients does not respond. In this study, we aimed to identify a blood-based immune marker associated with non-response to facilitate patient selection for anti-PD1. To this end, we quantified 18 immune cell populations using multiplex flow cytometry in blood samples from 71 patients with mUC (as part of a biomarker discovery trial; NCT03263039, registration date 28-08-2017). Patients were classified as responder (ongoing complete or partial response, or stable disease; n = 25) or non-responder (progressive disease; n = 46) according to RECIST v1.1 at 6 months of treatment with pembrolizumab. We observed no differences in numbers of lymphocytes, T-cells, granulocytes, monocytes or their subsets between responders and non-responders at baseline. In contrast, analysis of ratios of immune cell populations revealed that a high mature neutrophil-to-T-cell ratio (MNTR) exclusively identified non-responders. In addition, the survival of patients with high versus low MNTR was poor: median overall survival (OS) 2.2 vs 8.9 months (hazard ratio (HR) 6.6; p p

Purpose: PD-1 inhibition results in durable antitumor responses in a proportion of patients with metastatic urothelial cancer (mUC). The majority of patients, however, do not experience clinical benefit. In this study, we aimed to identify early changes in T-cell subsets that underlie anti–PD-1 efficacy in patients with mUC. Experimental Design: Paired samples were collected from peripheral blood, plasma, and metastatic lesions of 56 patients with mUC at baseline and weeks 6 and 12 after initiating pembrolizumab treatment (200 mg intravenously, every 3 weeks). Samples were analyzed using multiplex flow cytometry, ELISA, and in situ stainings, including cellular network analysis. Treatment response was evaluated as best overall response according to RECIST v1.1, and patients were classified as responder (complete or partial response) or nonresponder (progressive disease). Results: In responders, baseline fractions of CD4+ T cells expressing cosignaling receptors were higher compared with nonresponders. The fraction of circulating PD-1+ CD4+ T cells decreased at weeks 6 and 12, whereas the fraction of 4-1BB+ CD28+ CD4+ T cells increased at week 12. In metastatic lesions of responders, the baseline density of T helper-type 1 (Th1) cells, defined as T-bet+ CD4+ T cells, was higher as compared to non-responders. Upon treatment, Th1 cells became localized in close proximity to CD8+ T cells, CD11b+ myeloid cells, and tumor cells. Conclusions: A decrease in the fraction of circulating PD-1+ CD4+ T cells, and juxtaposition of Th1, CD8+, and myeloid cells was associated with response to anti–PD-1 treatment in patients with mUC.

Only a subgroup of triple-negative breast cancer (TNBC) responds to immune checkpoint inhibitors (ICI). To better understand lack of response to ICI, we analyze 681 TNBCs for spatial immune cell contextures in relation to clinical outcomes and pathways of T cell evasion. Excluded, ignored and inflamed phenotypes can be captured by a gene classifier that predicts prognosis of various cancers as well as anti-PD1 response of metastatic TNBC patients in a phase II trial. The excluded phenotype, which is associated with resistance to anti-PD1, demonstrates deposits of collagen-10, enhanced glycolysis, and activation of TGFβ/VEGF pathways; the ignored phenotype, also associated with resistance to anti-PD1, shows either high density of CD163+ myeloid cells or activation of WNT/PPARγ pathways; whereas the inflamed phenotype, which is associated with response to anti-PD1, revealed necrosis, high density of CLEC9A+ dendritic cells, high TCR clonality independent of neo-antigens, and enhanced expression of T cell co-inhibitory receptors., Only a subset of triple negative breast cancer patients respond to immunotherapy. Here, the authors analysed spatial immune contextures, which can be captured by a gene classifier, in relation to genomic alterations, mechanisms of T cell evasion and response to anti-PD1 treatment.

Multiple myeloma (MM) is characterized by loss of anti-tumor T cell immunity. Despite moderate success of treatment with anti-PD1 antibodies, effective treatment is still challenged by poor T cell-mediated control of MM. To better enable identification of shortcomings in T-cell immunity that relate to overall survival (OS), we interrogated transcriptomic data of bone marrow samples from eight clinical trials (n = 1654) and one trial-independent patient cohort (n = 718) for multivariate analysis. Gene expression of V-domain Ig suppressor of T cell activation (VISTA) was observed to correlate to OS [hazard ratio (HR): 0.72, 95% CI: 0.61–0.83, p = 0.005]. Upon imaging the immune contexture of MM bone marrow tissues (n = 22) via multiplex in situ stainings, we demonstrated that VISTA was expressed predominantly by CD11b+ myeloid cells. The combination of abundance of VISTA+, CD11b+ cells in the tumor but not stromal tissue together with low presence of CD8+ T cells in the same tissue compartment, termed a high VISTA-associated T cell exclusion score, was significantly associated with short OS [HR: 16.6, 95% CI: 4.54–62.50, p &lt, 0.0001]. Taken together, the prognostic value of a combined score of VISTA+, CD11b+ and CD8+ cells in the tumor compartment could potentially be utilized to guide stratification of MM patients for immune therapies.

In oral-cancer, the number of tumor-infiltrating lymphocytes (TILs) associates with improved survival, yet the prognostic value of the cellular composition and localization of TILs is not defined. We quantified densities, localizations, and cellular networks of lymphocyte populations in 138 patients with T1-T2 primary oral-tongue squamous cell carcinoma treated with surgical resections without any perioperative (chemo)radiotherapy, and correlated outcomes to overall survival (OS). Multiplexed in-situ immunofluorescence was performed for DAPI, CD4, CD8, CD20, and pan-cytokeratin using formalin-fixed paraffin-embedded sections, and spatial distributions of lymphocyte populations were assessed in the tumor and stroma compartments at the invasive margin (IM) as well as the center of tumors. We observed a high density of CD4, CD8, and CD20 cells in the stroma compartment at the IM, but neither lymphocyte densities nor networks as single parameters associated with OS. In contrast, assessment of two contextual parameters within the stroma IM region of tumors, i.e., the number of CD20 cells within 20 µm radii of CD20 and CD4 cells, termed the CD20 Cluster Score, yielded a highly significant association with OS (HR 0.38; p = .003). Notably, the CD20 Cluster Score significantly correlated with better OS and disease-free survival in multivariate analysis (HR 0.34 and 0.47; p = .001 and 0.019) as well as with lower local recurrence rate (OR: 0.13; p = .028). Taken together, our study showed that the presence of stromal B-cell clusters at IM, in the co-presence of CD4 T-cells, associates with good prognosis in early oral-tongue cancer patients.

Background Checkpoint inhibitors have become standard care of treatment for non-small cell lung cancer (NSCLC), yet only a limited fraction of patients experiences durable clinical benefit, highlighting the need for markers to stratify patient populations. Methods To prospectively identify patients showing response to therapy, we have stained peripheral blood samples of NSCLC patients treated with 2nd line nivolumab (n = 71), as well as healthy controls, with multiplex flow cytometry. By doing so, we enumerated 18 immune cell subsets and assessed expression for 28 T cell markers, which was followed by dimensionality reduction as well as rationale-based analyses. Results In patients with a partial response (PR), representing best overall response (BOR) according to RECIST v1.1, the number of CD8 T cells at baseline and during treatment is similar to those of healthy controls, but 2-fold higher than in patients with progressive and stable disease (PD and SD). CD8 T cell populations in PR patients show enhanced frequencies of T effector memory re-expressing CD45RA (TEMRA) cells, as well as T cells that express markers of terminal differentiation (CD95+) and egression from tumor tissue (CD69-). In PR patients, the fraction of CD8 T cells that lacks co-stimulatory receptors (CD28, ICOS, CD40L, 4-1BB, OX40) correlates significantly with the total numbers and differentiated phenotype of CD8 T cells. Conclusions This study demonstrates that high numbers of peripheral CD8 T cells expressing differentiation markers and lacking co-stimulatory receptors at baseline are associated with response to nivolumab in NSCLC patients. Electronic supplementary material The online version of this article (10.1186/s40425-019-0608-y) contains supplementary material, which is available to authorized users.

Cell survival, differentiation, and migration are all dependent on the cell’s interaction with its external environment. In addition to chemical cues, cells react to their physical environment, particularly the stiffness of the substrate. In order for cells to react to these elements, they must make use of cellular machinery to signal changes in their microenvironment. One such proposed machinery is the protein p130Cas, which has been shown to regulate focal adhesion turnover, actin dynamics, and cell migration. Here we show that p130Cas localizes to focal adhesions depending on substrate stiffness and subsequently modulates cellular force exertion. We compared on substrates of tunable stiffness knock-out CAS-/-cells to cells re-expressing either the full-length p130Cas or a mutant lacking the focal adhesion targeting domains. On polyacrylamide gels, we observed that p130Cas prevented focal adhesion formation at low stiffness. On structured micro-pillar arrays, p130Cas preferentially localized to sites of force exertion when the apparent Young’s modulus of the substrate was higher than E = 47 kPa. Stiffness-dependent localization of p130Cas coincided with slower, but increased force exertion for the full-length p130Cas. Cas localization to focal adhesions preceded force build-up by three minutes, suggesting a coordinating role for p130Cas in the cellular mechanoresponse. Thus, p130Cas appears to relay mechanosensory information in the cell through its ability to tune force exertion at the focal adhesion.

We investigate the geometrical and mechanical properties of adherent cells characterized by a highly anisotropic actin cytoskeleton. Using a combination of theoretical work and experiments on micropillar arrays, we demonstrate that the shape of the cell edge is accurately described by elliptical arcs, whose eccentricity expresses the degree of anisotropy of the internal cell stresses. This results in a spatially varying tension along the cell edge, that significantly affects the traction forces exerted by the cell on the substrate. Our work highlights the strong interplay between cell mechanics and geometry and paves the way towards the reconstruction of cellular forces from geometrical data., 5 pages, 3 figures

Tumor angiogenesis promotes tumor growth and metastasis. Here, we use automated sequential microprinting of tumor and endothelial cells in extracellular matrix (ECM) scaffolds to study its mechanical aspects. Quantitative reflection microscopy shows that tumor spheroids induce radial orientation of the surrounding collagen fiber network up to a distance of five times their radius. Across a panel of ~20 different human tumor cell lines, remote collagen orientation is correlated with local tumor cell migration behavior. Tumor induced collagen orientation requires contractility but is remarkably resistant to depletion of collagen-binding integrins. Microvascular endothelial cells undergo directional migration towards tumor spheroids once they are within the tumor-oriented collagen fiber network. Laser ablation experiments indicate that an intact physical connection of the oriented network with the tumor spheroid is required for mechanical sensing by the endothelial cells. Together our findings indicate that, in conjunction with described activities of soluble angiogenic factors, remote physical manipulation of the ECM network by the tumor can help to steer angiogenesis.

Cancer cells migrate from the primary tumour into surrounding tissue in order to form metastasis. Cell migration is a highly complex process, which requires continuous remodelling and re-organization of the cytoskeleton and cell-matrix adhesions. Here, we aimed to identify genes controlling aspects of tumour cell migration, including the dynamic organization of cell-matrix adhesions and cellular traction forces. In a siRNA screen targeting most cell adhesion-related genes we identified 200+ genes that regulate size and/or dynamics of cell-matrix adhesions in MCF7 breast cancer cells. In a subsequent secondary screen, the 64 most effective genes were evaluated for growth factor-induced cell migration and validated by tertiary RNAi pool deconvolution experiments. Four validated hits showed significantly enlarged adhesions accompanied by reduced cell migration upon siRNA-mediated knockdown. Furthermore, loss of PPP1R12B, HIPK3 or RAC2 caused cells to exert higher traction forces, as determined by traction force microscopy with elastomeric micropillar post arrays, and led to considerably reduced force turnover. Altogether, we identified genes that co-regulate cell-matrix adhesion dynamics and traction force turnover, thereby modulating overall motility behaviour.

Cells actively sense and process mechanical information that is provided by the extracellular environment to make decisions about growth, motility and differentiation. It is important to understand the underlying mechanisms given that deregulation of the mechanical properties of the extracellular matrix (ECM) is implicated in various diseases, such as cancer and fibrosis. Moreover, matrix mechanics can be exploited to program stem cell differentiation for organ-on-chip and regenerative medicine applications. Mechanobiology is an emerging multidisciplinary field that encompasses cell and developmental biology, bioengineering and biophysics. Here we provide an introductory overview of the key players important to cellular mechanobiology, taking a biophysical perspective and focusing on a comparison between flat versus three dimensional substrates. This article is part of a Special Issue entitled: Mechanobiology.

Integrin adhesion receptors connect the extracellular matrix (ECM) to the cytoskeleton and serve as bidirectional mechanotransducers. During development, angiogenesis, wound healing and cancer progression, the relative abundance of fibronectin receptors, including integrins α5β1 and αvβ3, changes, thus altering the integrin composition of cell–matrix adhesions. Here, we show that enhanced αvβ3 expression can fully compensate for loss of α5β1 and other β1 integrins to support outside-in and inside-out force transmission. α5β1 and αvβ3 each mediate actin cytoskeletal remodeling in response to stiffening or cyclic stretching of the ECM. Likewise, α5β1 and αvβ3 support cellular traction forces of comparable magnitudes and similarly increase these forces in response to ECM stiffening. However, cells using αvβ3 respond to lower stiffness ranges, reorganize their actin cytoskeleton more substantially in response to stretch, and show more randomly oriented traction forces. Centripetal traction force orientation requires long stress fibers that are formed through the action of Rho kinase (ROCK) and myosin II, and that are supported by α5β1. Thus, altering the relative abundance of fibronectin-binding integrins in cell–matrix adhesions affects the spatiotemporal organization of force transmission.

Cytoskeletal actin assemblies transmit mechanical stresses that molecular sensors transduce into biochemical signals to trigger cytoskeletal remodeling and other downstream events. How mechanical and biochemical signaling cooperate to orchestrate complex remodeling tasks has not been elucidated. Here, we studied remodeling of contractile actomyosin stress fibers. When fibers spontaneously fractured, they recoiled and disassembled actin synchronously. The disassembly rate was accelerated more than twofold above the resting value, but only when contraction increased the actin density to a threshold value following a time delay. A mathematical model explained this as originating in the increased overlap of actin filaments produced by myosin II-driven contraction. Above a threshold overlap, this mechanical signal is transduced into accelerated disassembly by a mechanism that may sense overlap directly or through associated elastic stresses. This biochemical response lowers the actin density, overlap, and stresses. The model showed that this feedback mechanism, together with rapid stress transmission along the actin bundle, spatiotemporally synchronizes actin disassembly and fiber contraction. Similar actin remodeling kinetics occurred in expanding or contracting intact stress fibers but over much longer timescales. The model accurately described these kinetics, with an almost identical value of the threshold overlap that accelerates disassembly. Finally, we measured resting stress fibers, for which the model predicts constant actin overlap that balances disassembly and assembly. The overlap was indeed regulated, with a value close to that predicted. Our results suggest that coordinated mechanical and biochemical signaling enables extended actomyosin assemblies to adapt dynamically to the mechanical stresses they convey and direct their own remodeling.

Item does not contain fulltext Interactions with the extracellular matrix (ECM) through integrin adhesion receptors provide cancer cells with physical and chemical cues that act together with growth factors to support survival and proliferation. Antagonists that target integrins containing the beta1 subunit inhibit tumor growth and sensitize cells to irradiation or cytotoxic chemotherapy in preclinical breast cancer models and are under clinical investigation. We found that the loss of beta1 integrins attenuated breast tumor growth but markedly enhanced tumor cell dissemination to the lungs. When cultured in three-dimensional ECM scaffolds, antibodies that blocked beta1 integrin function or knockdown of beta1 switched the migratory behavior of human and mouse E-cadherin-positive triple-negative breast cancer (TNBC) cells from collective to single cell movement. This switch involved activation of the transforming growth factor-beta (TGFbeta) signaling network that led to a shift in the balance between miR-200 microRNAs and the transcription factor zinc finger E-box-binding homeobox 2 (ZEB2), resulting in suppressed transcription of the gene encoding E-cadherin. Reducing the abundance of a TGFbeta receptor, restoring the ZEB/miR-200 balance, or increasing the abundance of E-cadherin reestablished cohesion in beta1 integrin-deficient cells and reduced dissemination to the lungs without affecting growth of the primary tumor. These findings reveal that beta1 integrins control a signaling network that promotes an epithelial phenotype and suppresses dissemination and indicate that targeting beta1 integrins may have undesirable effects in TNBC.

The mechanical respons of the micro environment of a cell is of great influence for its differentiation and mobility. However, the mechanism that a cell uses to sense these mechanical properties remains largely unkown. Mechanosensing could happen by direct coupling to the nucleus via the actin cytoskeleton or by activating molecular signaling cascades through protein complexes.A recent combination of traction force microscopy with super resolution imaging allowed for novel research into the nanoscale architecture of force-bearing focal adhesions. Here we apply this technique to investigate the relation between local force exertion by the cell and the number of signal transduction proteins inside the integrin adhesome.Integrin adhesomes are mechanosensory protein complexes that couple the intracelular force bearing actomyosin structures to the extracelular environment. These complexes contain signal transduction proteins like paxillin, vinculin, talin and FAK, that change conformation when force is exerted on the complex.By immunostaining these proteins with Alexa647 their position can be localized to 20nm in fixed cells. However, due to the photophysical properties of Alexa647, and the binding stoichiometry in antibody staining the number of localizations does not scale linearly with the number of signal transduction proteins. To overcome this problem we present a novel analysis method to relate the number of localizations to the minimal number of signal transduction proteins.

Chemical and physical cues from the extracellular matrix (ECM) are used as input for an integrated mechanochemical sensory system that controls cell behavior. Forces derived from the ECM have been implicated in various biological systems, including mesenchymal stem cell differentiation and tumor growth. Integrin transmembrane receptors connect the ECM network to the intracellular actomyosin network and are expected to play an important role in force sensing. Our group has previously shown that switching between two different integrin heterodimers ([alpha]5[beta]1 vs [alpha]v[beta]3), that bind the same ECM protein (fibronectin), causes remarkably distinct actin cytoskeletal organization. To investigate the role of these integrins in cellular mechanotransduction, we used fibronectin coated polyacrylamide substrates with varying rigidities and micropillar arrays. Data obtained thus far point to distinct rigidity thresholds for spreading and different distribution of ECM pulling forces for cells expressing either of these integrins. In order to further unravel the role of these integrins in cellular mechanotransduction we developed cyclic substrate stretcher technology coupled to real time confocal imaging.

Interactions with the extracellular matrix (ECM) provide cells with physical and chemical cues that act in concert with growth factors to support survival and proliferation. Transmembrane receptors of the integrin family mediate ECM attachment and play important roles in sensing and responding to ECM properties. Integrin signaling involves large integrin-associated intracellular protein complexes that act as anchors for the cytoskeleton and as signaling hotspots where enzymes and substrates are concentrated. Moreover, many different growth factor signaling cascades are amplified when cells are attached to the ECM. Integrins are involved in many pathologies; here we focus on their roles in cancer. Although "anchorage-independence" is a hallmark of cancer cells, genetic studies clearly show that integrins and associated proteins provide essential support for early tumor development and growth. Integrins also provide support during later stages of tumor progression but in some scenarios they appear to have suppressive activity, which is currently not understood.

Cells assemble a variety of bundled actomyosin structures in the cytoskeleton for activities such as cell-shape regulation, force production, and cytokinesis. Although these linear structures exhibit varied architecture, two common organizational themes are a punctate distribution of myosin II and distinct patterns of actin polarity. The mechanisms that cells use to assemble and maintain these organizational features are poorly understood. To study these, we reconstituted actomyosin bundles in vitro that contained only actin filaments and myosin II. Upon addition of ATP, the bundles contracted and the uniformly distributed myosin spontaneously reorganized into discrete clusters. We developed a mathematical model in which the motion of myosin II filaments is governed by the polarities of the actin filaments with which they interact. The model showed that the assembly of myosins into clusters is driven by their tendency to migrate to locations with zero net actin filament polarity. With no fitting parameters, the predicted distribution of myosin cluster separations was in close agreement with our experiments, including a −3/2 power law decay for intermediate length scales. Thus, without an organizing template or accessory proteins, a minimal bundle of actin and myosin has the inherent capacity to self-organize into a heterogeneous banded structure.