Biology subjects

Irianto, J.

Publications and source records attributed to Irianto, J..

16 recordsLinked to original sources

bioRxiv2024

Glioblastoma invasion relies on actomyosin contractility and metalloproteinase activity

PurposeGlioblastoma multiforme (GBM) is an aggressive brain tumor with a 5-year survival rate below 7%. Poor outcomes are driven in part by diffuse invasion into surrounding brain tissue, which limits complete surgical resection and promotes recurrence. Improved therapies require a quantitative understanding of the mechanics of GBM invasion. MethodsPatient-derived GBM neurospheres were embedded in Matrigel and analyzed using live imaging, three-dimensional traction force microscopy (3D TFM), and targeted cytoskeletal perturbations. ResultsInvading GBM cells adopted an elongated, protrusion-rich morphology, with F-actin enriched at the cell periphery and microtubules extending along the protrusion shaft, consistent with a mesenchymal-like invasion program. 3D TFM revealed sustained matrix engagement, including progressive bead clustering, increasing cumulative traction forces, and traction hotspots concentrated near protrusion tips. Perturbation studies separated cytoskeletal requirements for invasion and force transmission: actin polymerization was essential for invasion, myosin II activity was required for robust traction generation and efficient invasion, and microtubule polymerization supported directional persistence and maximal traction output. Notably, a low level of invasion persisted under myosin II inhibition despite minimal detectable traction forces, and this residual invasion was not suppressed by pan-MMP inhibition, indicating a traction-poor, MMP-independent invasion component in Matrigel. ConclusionsThese findings establish a quantitative mechanical framework for GBM neurosphere invasion in 3D Matrigel and define distinct contributions of actomyosin contractility and microtubules to invasive progression.

cancer biology↗

bioRxiv2024

Rotational migration in human pancreatic ductal organoids depends on actin and myosin activity

Rotational migration is one specific form of collective cell migration when epithelial cells are confined in a spherical geometry, such as in the epithelial acini. This tissue-level rotation motion is crucial for the morphogenesis of multiple epithelial systems. Here, we introduce a new primary human model for the study of rotational migration, pancreatic ductal organoids. Live imaging revealed the persistent rotation of the organoids over time. By tracking the nuclei, the three-dimensional trajectory of the cellular movement was reconstructed and the velocity of the rotation was quantified. The presence of focal adhesion clusters and prominent actin stress fibers were observed at the basal side of the organoids, suggesting the interactions between the cells and the surrounding extracellular matrix. Finally, our inhibition study showed the dependence of pancreatic ductal organoid rotational migration on myosin activity, actin polymerization, and actin branching. We hope that this model will enable future studies with human primary cells, which are more faithful to normal epithelial cells.

cell biology↗

bioRxiv2023

Confinement with Myosin-II suppression increases heritable loss of chromosomes, using live-cell ChReporters

Matrix around cells exerts many effects, some of which depend on the putative tumor suppressor Myosin-II, but whether such factors affect DNA sequences in a cell remains unclear. Here, live-cell monitoring of changes to chromosome copy numbers is developed and studied under diverse perturbations, including Myosin-II inhibition in confined mitosis. Squeezing of mitotic cells is seen in vivo and kills in vitro, but stem cells and cancer cells that survive show heritable loss of mono-allelic GFP/RFP-tagged constitutive genes that function as novel Chromosome-reporters (ChReporters). Myosin-II suppression increases such loss in 3D & 2D confinement but not in standard 2D, with "lethal" multipolar divisions proving myosin-dependent. Viable chromosome loss after confined mitosis associates more with mis-segregation than with multipolars or division number. Solid human tumors and teratomas in mice also show ChReporter loss and a confinement-signature of Myosin-II suppression, although losses are selected against in 2D culture. Heritable loss in rigid-confinement also appears independent of a spindle assembly checkpoint that functions in 2D. Confinement and myosin-II thus regulate pathways of heritable mechanogenetic change.

cell biology↗

bioRxiv2022

Differential roles of extracellular matrix proteins on primary and metastatic pancreatic cancer cells

Pancreatic cancer 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 non-malignant 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.

cancer biology↗

bioRxiv2022

Genomic variations drive phenotypic heterogeneity in pancreatic cancer cells

ObjectivesTo evaluate the suitability of the MIA PaCa-2 cell line for studying pancreatic cancer intratumor heterogeneity, we aim to further characterize the nature of MIA PaCa-2 cells phenotypic, genomic, and transcriptomic heterogeneity. MethodsMIA PaCa-2 single-cell clones were established through flow cytometry. For the phenotypic study, we quantified the cellular morphology, proliferation rate, migration potential, and drug sensitivity of the clones. The chromosome copy number and transcriptomic profiles were quantified using SNPa and RNA-seq, respectively. ResultsFour MIA PaCa-2 clones showed distinctive phenotypes, with differences in cellular morphology, proliferation rate, migration potential, and drug sensitivity. We also observed a degree of genomic variations between these clones in form of chromosome copy number alterations and single nucleotide variations, suggesting the genomic heterogeneity of the population, and the intrinsic genomic instability of MIA PaCa-2 cells. Lastly, transcriptomic analysis of the clones also revealed gene expression profile differences between the clones, including the uniquely regulated ITGAV, which dictates the morphology of MIA PaCa-2 clones. ConclusionsMIA PaCa-2 is comprised of cells with distinctive phenotypes, heterogeneous genomes, and differential transcriptomic profiles, suggesting its suitability as a model to study the underlying mechanisms behind pancreatic cancer heterogeneity.

cancer biology↗

bioRxiv2022

SCsnvcna: Integrating SNVs and CNAs on a phylogenetic tree from single-cell DNA sequencing data

Single-cell DNA sequencing enables the construction of evolutionary trees that can reveal how tumors gain mutations and grow. Different whole genome amplification (WGA) procedures render genomic materials of different characteristics, often suitable for the detection of either single nucleotide variation (SNV) or for copy number aberration (CNA), but not for both, hindering the placement of both SNVs and CNAs on the same phylogenetic tree for the study of interplay of SNVs and CNAs. SCARLET places SNVs on a CNA tree, a tree derived based on the copy number profiles, while considering SNV loss due to copy number losses. However, SCARLET requires that the SNVs and CNAs are detected from the same sets of cells, which is technically challenging due to the sequencing errors or the low sequencing coverage associated with a particular WGA procedure. Here we presented a novel computational tool, SCsnvcna, that aims at placing SNVs on a CNA tree whereas the sets of cells rendering the SNVs and CNAs are independent, thus is more practical in terms of the technical challenge from single cell WGA process. SCsnvcna is a Bayesian probabilistic model that utilizes both the genotype constraints on the tree and the cellular prevalence (CP) to search the solution that has the highest joint probability. Both simulated and real datasets show that SCsnvcna is highly accurate in predicting the placement of SNVs and SNV cells. In addition, SCsnvcna has a precise prediction of SNV losses due to copy number loss.

bioinformatics↗

bioRxiv2022

Genomic heterogeneity in pancreatic cancer organoids and its stability with culture

The establishment of patient-derived pancreatic cancer organoid culture in recent years creates an exciting opportunity for researchers to perform a wide range of in vitro studies on a model that closely recapitulates the tumor. Among the outstanding questions in pancreatic cancer biology are the causes and consequences of genomic heterogeneity observed in the disease. However, to use pancreatic cancer organoids as a model to study genomic variations, we need to first understand the degree of genomic heterogeneity and its stability within organoids. Here, we used single-cell whole-genome sequencing to investigate the genomic heterogeneity of two independent pancreatic cancer organoids, as well as their genomic stability with extended culture. Clonal populations with similar copy number profiles were observed within the organoids, and the proportion of these clones was shifted with extended culture, suggesting the growth advantage of some clones. However, sub-clonal genomic heterogeneity was also observed within each clonal population, indicating the genomic instability of the pancreatic cancer cells themselves. Furthermore, our transcriptomic analysis also revealed a positive correlation between copy number alterations and gene expression regulation, suggesting the functionality of these copy number alterations.

cancer biology↗

bioRxiv2022

Cooperative phagocytosis underlies macrophage immunotherapy of solid tumours and initiates a broad anti-tumour IgG response

Macrophages are abundant in solid tumours and typically associate with poor prognosis, but macrophage clusters in tumour nests have also been reported as beneficial even though dispersed macrophages would have more contacts with cancer cells. Here, by maximizing both phagocytic activity and macrophage numbers, we discover cooperative phagocytosis by low entropy clusters in rapidly growing engineered immuno-tumouroids. The results fit the calculus of proliferation-versus-engulfment, and rheological measurements and molecular perturbations provide a basis for understanding phagocytic disruption of a tumours cohesive forces in soft cellular phases. The perturbations underscore the utility of suppressing a macrophage checkpoint in combination with an otherwise ineffective tumour-opsonizing monoclonal antibody, and the approach translates in vivo to tumour elimination that durably protects mice from re-challenge and metastasis. Adoptive transfer of engineered macrophages increases the fraction of mice that eliminate tumours and potentially overcomes checkpoint blockade challenges in solid tumours like insufficient permeation of blocking antibodies and on-target, off-tumour binding. Finally, anti-cancer IgG induced in vivo are tumour-specific but multi-epitope and contribute to a phagocytic feedback that drives macrophage clustering in vitro. Given that solid tumours remain challenging for immunotherapies, durable anti-tumour responses here illustrate unexpected advantages in maximizing net phagocytic activity.

bioengineering↗

bioRxiv2021

Scaling concepts in 'omics: nuclear lamin-B scales with tumor growth and predicts poor prognosis, whereas fibrosis can be pro-survival

Spatiotemporal relationships between genes expressed in tissues likely reflect physicochemical principles that range from stoichiometric interactions to co-organized fractals with characteristic scaling. For key structural factors within the nucleus and extracellular matrix (ECM), gene-gene power laws are found to be characteristic across several tumor types in The Cancer Genome Atlas (TCGA) and across single-cell RNA-seq data. The nuclear filament LMNB1 scales with many tumor-elevated proliferation genes that predict poor survival in liver cancer, and cell line experiments show LMNB1 regulates cancer cell cycle. Also high in the liver, lung, and breast tumors studied here are the main fibrosis-associated collagens, COL1A1 and COL1A2, that scale stoichiometrically with each other and super-stoichiometrically with a pan-cancer fibrosis gene set. However, high fibrosis predicts prolonged survival of patients undergoing therapy and does not correlate with LMNB1. Single-cell RNA-seq data also reveal scaling consistent with the pan-cancer power laws obtained from bulk tissue, allowing new power law relations to be predicted. Lastly, although noisy data frustrate weak scaling, concepts such as stoichiometric scaling highlight a simple, internal consistency check to qualify expression data. ClassificationApplied Physical Sciences (major) and Cell Biology (minor) Significance StatementNon-linear scaling analyses pervade polymer physics and chemistry and conceivably provide new insight into polymeric assemblies of genes expressed in tissues as well as co-regulated gene sets. Fractal scaling and stoichiometric scaling are among the gene-gene power law results identified here for key structural polymers in nuclei or extracellular matrix in human cancer data. Among nuclear envelope factors that might scale with DNA mass, only one nuclear filament scales with tumor proliferation and predicts poor survival in some cancer types. Collagen-1 scales with fibrosis and also tends to increase in multiple tumor types, but patients in therapy surprisingly survive longest with the highest levels of fibrosis, consistent with a therapeutic response.

cancer biology↗

bioRxiv2021

Heterogeneous strains in tissue collagen show that high strains locally suppress degradation by collagenase

Collagen, the most abundant protein in mammals, contributes to the physical properties of different tissues during development, homeostasis, and disease. The adaptation of physical properties of tissues to mechanical stimuli is thus dependent on the control of tissue collagen levels by well-regulated synthesis and degradation of collagen. Importantly, how various molecular-level events within a tissue sustaining a range of mechanical strains contribute towards maintaining its collagen levels, remains unclear to date. Such molecular level processes in tissues are studied here in the case of isolated tendons consisting of collagen fibrils oriented along tissue loading-axis and beating embryonic hearts to gain understanding of mechanical load dependent tissue sculpting. Using a novel bioreactor design, starved mice tail tendon fascicles were used as a "cell-free" model and were subjected to heterogeneous and uniaxial deformation modes. Patterned photobleaching of fluorescent probes, a novel Aza-peptide or dye, on fascicles used to quantify tissue strains. Tissue microstructure was simultaneously imaged using second harmonic generation (SHG) signal to assess tissue collagen content while deformed fascicle samples were exposed to purified matrix metalloproteinase-1 (MMP-1) or bacterial collagenase (BC). A decrease in the degradation rate (relative to strain-free) was observed for physiological strain limits of tendon tissue (i.e. [~]5-8%) while at higher strains (i.e. pathological) the degradation rate was independent of strain magnitude changes. Interestingly, the strain dependence of degradation rate was independent of cleavage-site specificity of the collagenase molecules and the mode of tendon tissue deformation. Although spatially different within a tissue sample, the values of strain, degradation rate and collagen fiber organization with time during degradation of each tendon fascicle region were highly correlated. Tendon regions dominated by collagen fibers inclined to fascicle-axis were observed to follow non-affine deformation. The dependence of the degradation rate on mechanical strain is due to sequestration of collagen cleavage sites within fibrils. Permeation, tissue mass density and mobility of fluorescent collagenase and dextran are strain-independent for fascicle strains up to [~]5-8% while the degradation rate is positively correlated to unfolded triple-helical collagen content. Normal beating chick hearts subjected to [~]5% peak strain in a spatiotemporal coordinate contractile wave were observed to maintain their collagen mass until the beating strain is suppressed by inhibition of myosin-II. Based on the presence of exogeneous MMP inhibitors, endogenous MMPs within the non-beating hearts degrade the collagens immediately (in [~]30-60 mins). Both tissue systems under mechanical strains suggest degradative sculpting where mechanical strain-dependent collagen fibril architecture changes appear to play a key role in determining collagen lifetime within tissues. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=139 HEIGHT=200 SRC="FIGDIR/small/430141v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@17da11borg.highwire.dtl.DTLVardef@dbfb8forg.highwire.dtl.DTLVardef@6069b9org.highwire.dtl.DTLVardef@1bd273e_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2019

Mechanosensing by the lamina protects against nuclear rupture, DNA damage, and cell cycle arrest

Whether cell forces or extracellular matrix (ECM) can impact genome integrity is largely unclear. Here, acute perturbations (~1hr) to actomyosin stress or ECM elasticity cause rapid and reversible changes in lamin-A, DNA damage, and cell cycle. Embryonic hearts, differentiated iPS-cells, and various nonmuscle cell types all show that actomyosin-driven nuclear rupture causes cytoplasmic mis-localization of DNA repair factors and excess DNA damage. Binucleation and micronuclei increase as telomeres shorten, which all favor cell cycle arrest. Deficiencies in lamin-A and repair factors exacerbate these effects, but lamin-A-associated defects are rescued by repair factor overexpression and by contractility modulators in clinical trials. Contractile cells on stiff ECM normally exhibit low phosphorylation and slow degradation of lamin-A by matrix-metalloprotease-2 (MMP2), and inhibition of this lamin-A turnover and also actomyosin contractility is seen to minimize DNA damage. Lamin-A is thus stress-stabilized to mechano-protect the genome.

cell biology↗

bioRxiv2018

Live cell monitoring for factors affecting genome variation

Cancer cells and pluripotent stem cells frequently exhibit gains or losses of entire chromosomes and chromosome segments, and the typical terminal analyses of genomes suggest this aneuploidy is ongoing and particularly variable in solid tumors. Here, we quantify aneuploidy-inducing perturbations by live cell fluorescence monitoring for changes in chromosome-5 in a lung cancer line and in normal diploid iPS cells. Inhibition of the spindle assembly checkpoint (SAC) and knockdown of DNA repair factors cause chromosome mis-segregation to increase several-fold above a low baseline level, and both perturbations also generate several-fold more rare fluorescent-null cells. Loss of chromosome-5 is confirmed by single cell karyotyping, SNP arrays on stable isolated clones, and downregulated expression of genes on chromosome-5 in single cell transcriptomics. The iPS cells also show loss of fluorescence in infrequent cells after SAC inhibition and upon growth as teratomas in mice. Viability, selection, and altered expression can thus be tracked to reveal molecular mechanisms in aneuploidy.

genomics↗

bioRxiv2018

Rescue of DNA damage in cells after constricted migration reveals bimodal mechano-regulation of cell cycle

Migration through constrictions can clearly rupture nuclei and mis-localize nuclear proteins but damage to DNA remains uncertain as does any effect on cell cycle. Here, myosin-II inhibition rescues rupture and partially rescues the DNA damage marker {gamma}H2AX, but an apparent delay in cell cycle is unaffected. Co-overexpression of multiple DNA repair factors and antioxidant inhibition of break formation also have partial effects, independent of rupture. However, there seems to be a bimodal dependence of cell cycle on DNA damage. Migration through custom-etched pores yields the same bimodal, with ~4-m pores causing intermediate levels of damage and cell cycle delay. Micronuclei (generated in faulty division) of the smallest diameter appear similar to ruptured nuclei, with high DNA damage and entry of chromatin-binding cGAS (cyclic-GMP-AMP-synthase) from cytoplasm but low repair factor levels. Increased genomic variation after constricted migration is quantified in expanding clones and is consistent with (mis)repair of excess DNA damage and subsequent proliferation.

cell biology↗

bioRxiv2017

Cell cycle repression and DNA repair defects follow constricted migration

Cancer cell invasion into tissue or narrow capillaries often elongates the nucleus and sometimes damages it, but cell cycle effects are unknown and highly relevant to tumorigenesis. Here, nuclear rupture and DNA breaks caused by constricted migration are quantified in different phases of cell cycle - which is effectively repressed. Cancer lines with varying levels of contact inhibition and lamina proteins exhibit diverse frequencies of nuclear lamina rupture after migration, with prerupture dilation of gene-edited RFP-Lamin-B1 preceding DNA repair factor leakage in pressure-controlled distension. Post-migration rupture indeed associates with mis-localized DNA repair factors and increased DNA breaks as quantified by pan-nucleoplasmic foci of {gamma}H2AX, with foci counts always suppressed in late cell cycle. When contact-inhibited cells migrate through large pores into sparse microenvironments, cells re-enter cell cycle consistent with release from contact inhibition. In contrast, constricting pores effectively delay re-entry, but the excess DNA damage nonetheless exceeds any cell cycle dependence. Partial depletion of topoisomerase does not strongly affect cell cycle or the excess DNA damage, consistent with weak dependencies on replication stress. Constricted migration thus impacts cell cycle as well as DNA damage.

biophysics↗

bioRxiv2017

Mitotic progression following DNA damage enables pattern recognition within micronuclei

Inflammatory gene expression following genotoxic cancer therapy is well documented, yet the events underlying its induction remain poorly understood. Inflammatory cytokines modify the tumor microenvironment by recruiting immune cells and are critical for both local and systemic (abscopal) tumor responses to radiotherapy1. An enigmatic feature of this phenomenon is its delayed onset (days), in contrast to the acute DNA damage responses that occur in minutes to hours. Such dichotomous kinetics implicate additional rate limiting steps that are essential for DNA-damage induced inflammation. Here, we show that cell cycle progression through mitosis following DNA double-strand breaks (DSBs) leads to the formation of micronuclei, which precede activation of inflammatory signaling and are a repository for the pattern recognition receptor cGAS. Inhibiting progression through mitosis or loss of pattern recognition by cGAS-STING impaired interferon signaling and prevented the regression of abscopal tumors in the context of ionizing radiation and immune checkpoint blockade in vivo. These findings implicate temporal modulation of the cell cycle as an important consideration in the context of therapeutic strategies that combine genotoxic agents with immune checkpoint blockade.

cancer biology↗

bioRxiv2016

DNA damage in 3D constricted migration or after lamin-A depletion in 2D: shared mechanisms of repair factor mis-localization under nuclear stress

Cells that migrate through small, rigid pores and that have normal levels of the nuclear structure protein lamin-A exhibit an increase in DNA damage, which is also observed with lamin-A depletion in diseases such as cancer and with many lamin-A mutations. Here we show nuclear envelope rupture is a shared feature that increases in standard culture after lamin-A knockdown, which causes nuclear loss of multiple DNA repair factors and increased DNA damage. Some repair factors are merely mis-localized to cytoplasm whereas others are partially depleted unless rescued by lamin-A expression. Compared to standard cultures on rigid glass coverslips, the growth of lamin-A low cells on soft matrix relaxes cytoskeletal stress on the nucleus, suppresses the mis-localization of DNA repair factors, and minimizes DNA damage nearly to wildtype levels. Conversely, constricted migration of the lamin-A low cells causes abnormally high levels of DNA damage, consistent with sustained loss of repair factors. The findings add insight into why monogenic progeroid syndromes that often associate with increased DNA damage and predominantly impact cells in stiff tissues result from mutations only in lamin-A or DNA repair factors.

cell biology↗