Intercellular Dynamics Research Articles

Advances, challenges and future applications of liver organoids in experimental regenerative medicine

The liver is a vital organ responsible for numerous metabolic processes in the human body, including the metabolism of drugs and nutrients. After liver damage, the organ can rapidly return to its original size if the causative factor is promptly eliminated. However, when the harmful stimulus persists, the liver’s regenerative capacity becomes compromised. Substantial theoretical feasibility has been demonstrated at the levels of gene expression, molecular interactions, and intercellular dynamics, complemented by numerous successful animal studies. However, a robust model and carrier that closely resemble human physiology are still lacking for translating these theories into practice. The potential for liver regeneration has been a central focus of ongoing research. Over the past decade, the advent of organoid technology has provided improved models and materials for advancing research efforts. Liver organoid technology represents a novel in vitro culture system. After several years of refinement, human liver organoids can now accurately replicate the liver’s morphological structure, nutrient and drug metabolism, gene expression, and secretory functions, providing a robust model for liver disease research. Regenerative medicine aims to replicate human organ or tissue functions to repair or replace damaged tissues, restore their structure or function, or stimulate the regeneration of tissues or organs within the body. Liver organoids possess the same structure and function as liver tissue, offering the potential to serve as a viable replacement for the liver, aligning with the goals of regenerative medicine. This review examines the role of liver organoids in regenerative medicine.

Abstract B022: Investigating how monocytes impair NK cell cytotoxicity in lung metastasis of triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) accounts for 20% of breast cancer (BC) cases worldwide, yet due to its aggressive phenotype and lack of targeted therapies it has the worst prognosis amongst BC subtypes. Up to 40% of TNBC patients will relapse after treatment and develop distant metastasis. Metastasis is the complex process by which disseminated tumor cells (DTCs) leave the primary tumor and grow at distal sites. Since most metastases occur within 3 years of diagnosis, the rapid onset and lack of effective treatments in metastatic TNBC (mTNBC) means less than 20% of patients survive after 4 years. Even though it is the most “immune-activated” BC subtype, and immune checkpoint blockade (ICB) has shown clinical benefits in early TNBC, there is a clear need for novel immunotherapeutic approaches for patients with mTNBC. Natural killer (NK) cells are innate lymphocytes that function as the body’s first line defense against metastatic cancer cells. NK cells eliminate DTCs in the blood or at distal sites via NK cell cytotoxicity (NKCC) and this process is known to be impaired in successful metastasis. We hypothesized that other cells within the tumor microenvironment (TME) can inhibit NKCC at the earliest stages of metastatic outgrowth. We have used Cherry-niche, an in vivo labelling tool, to characterize how TME cells change in early and established lung micro-metastases by single-cell RNA sequencing (scRNA-seq) and flow cytometry. Our initial analyses showed that monocytes are highly enriched, not only in the entire metastatic lung, but specifically in the established niche surrounding DTCs. Although we detected a slight increase in total NK cells, there was a shift in maturation status with fewer mature NK cells (CD27-CD11b+) and more immature NK cells (CD27+/-CD11b-) in the metastatic lung. Furthermore, NK cells were largely absent from the metastatic niche and those that were present displayed a dysfunctional phenotype. CellChat and NicheNet analyses of our scRNA-seq data predicted that several receptor-ligand interactions, including the cytokine macrophage migration inhibitory factor (MIF), can mediate the crosstalk between NK cells and monocytes. Next, we developed an in vitro 3D co-culture system to study the effect of blood-derived monocytes on human NKCC against mTNBC cells. We found that monocytes can suppress NKCC against MDA-MB-231 cells and decrease the viability of mature NK cells. Furthermore, recombinant MIF was able to directly inhibit NKCC, whilst a selective MIF antagonist was able to rescue monocyte-induced impairment of NKCC. These data implicate MIF as a critical monocyte-derived factor in the suppression of NK cell function against mTNBC cells. Future work will aim to identify the exact mechanism by which MIF inhibits NKCC, whilst also evaluate the effect of monocyte depletion and MIF inhibition on metastatic outgrowth and NK cell activation/maturation in vivo. This study will reveal novel inter-cellular dynamics occurring in the metastatic lung, as well as immunotherapeutic targets that may restore NKCC in mTNBC. Citation Format: Christos Ermogenous, Luigi Ombrato, Gordon Beattie. Investigating how monocytes impair NK cell cytotoxicity in lung metastasis of triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B022.

Single-cell analysis of nasal epithelial cell development in domestic pigs

The nasal mucosa forms a critical barrier against the invasion of respiratory pathogens. Composed of a heterogeneous assortment of cell types, the nasal mucosa relies on the unique characteristics and complex intercellular dynamics of these cells to maintain their structural integrity and functional efficacy. In this study, single-cell RNA sequencing (scRNA-seq) of porcine nasal mucosa was performed, and nineteen distinct nasal cell types, including nine epithelial cell types, five stromal cell types, and five immune cell types, were identified. The distribution patterns of three representative types of epithelial cells (basal cells, goblet cells, and ciliated cells) were subsequently detected by immunofluorescence. We conducted a comparative analysis of these data with published human single-cell data, revealing consistent differentiation trajectories among porcine and human nasal epithelial cells. Specifically, basal cells serve as the initial stage in the differentiation process of nasal epithelial cells, which then epithelial cells. This research not only enhances our understanding of the composition and transcriptional signature of porcine nasal mucosal cells but also offers a theoretical foundation for developing alternative models for human respiratory diseases.

Long-term mesoscale imaging of 3D intercellular dynamics across a mammalian organ

A comprehensive understanding of physio-pathological processes necessitates non-invasive intravital three-dimensional (3D) imaging over varying spatial and temporal scales. However, huge data throughput, optical heterogeneity, surface irregularity, and phototoxicity pose great challenges, leading to an inevitable trade-off between volume size, resolution, speed, sample health, and system complexity. Here, we introduce a compact real-time, ultra-large-scale, high-resolution 3D mesoscope (RUSH3D), achieving uniform resolutions of 2.6×2.6×6μm3 across a volume of 8,000×6,000×400μm3 at 20Hz with low phototoxicity. Through the integration of multiple computational imaging techniques, RUSH3D facilitates a 13-fold improvement in data throughput and an orders-of-magnitude reduction in system size and cost. With these advantages, we observed premovement neural activity and cross-day visual representational drift across the mouse cortex, the formation and progression of multiple germinal centers in mouse inguinal lymph nodes, and heterogeneous immune responses following traumatic brain injury-all at single-cell resolution, opening up a horizon for intravital mesoscale study of large-scale intercellular interactions at the organ level.

Investigation of cellular communication and signaling pathways in tumor microenvironment for high TP53-expressing osteosarcoma cells through single-cell RNA sequencing.

Osteosarcoma (OS) stands as the most prevalent primary bone cancer in children and adolescents, and its limited treatment options often result in unsatisfactory outcomes, particularly for metastatic cases. The tumor microenvironment (TME) has been recognized as a crucial determinant in OS progression. However, the intercellular dynamics between high TP53-expressing OS cells and neighboring cell types within the TME are yet to be thoroughly understood. In our study, we harnessed the single-cell RNA sequencing (scRNA-seq) technology in combination with the computational tool-Cellchat, aiming to elucidate the intercellular communication networks present within OS. Through meticulous quantitative inference and subsequent analysis of these networks, we succeeded in identifying significant signaling pathways connecting high TP53-expressing OS cells with proximate cell types, namely Macrophages, Monocytes, Endothelial Cells, and PVLs. This research brings forth a nuanced understanding of the intricate patterns and coordination involved in the TME's intercellular communication signals. These findings not only provide profound insights into the molecular mechanisms underpinning OS but also indicate potential therapeutic targets that could revolutionize treatment strategies.

Differential response of placental cells to high D‐glucose and its impact on extracellular vesicle biogenesis and trafficking via small GTPase Ras‐related protein RAB‐7A

AbstractPlacental extracellular vesicles (EVs) can be found in the maternal circulation throughout gestation, and their concentration, content and bioactivity are associated with pregnancy outcomes, including gestational diabetes mellitus (GDM). However, the effect of changes in the maternal microenvironment on the mechanisms associated with the secretion of EVs from placental cells remains to be fully established. Here, we evaluated the effect of high glucose on proteins associated with the trafficking and release of different populations of EVs from placental cells. BeWo and HTR8/SVneo cells were used as placental models and cultured under 5‐mM D‐glucose (i.e. control) or 25‐mM D‐glucose (high glucose). Cell‐conditioned media (CCM) and cell lysate were collected after 48 h. Different populations of EVs were isolated from CCM by ultracentrifugation (i.e. pellet 2K‐g, pellet 10K‐g, and pellet 100K‐g) and characterised by Nanoparticle Tracking Analysis. Quantitative proteomic analysis (IDA/SWATH) and multiple reaction monitoring protocols at high resolution (MRMHR) were developed to quantify 37 proteins related to biogenesis, trafficking/release and recognition/uptake of EVs. High glucose increased the secretion of total EVs across the pellets from BeWo cells, an effect driven mainly by changes in the small EVs concentration in the CCM. Interestingly, no effect of high glucose on HTR8/SVneo cells EVs secretion was observed. High glucose induces changes in proteins associated with vesicle trafficking in BeWo cells, including Heat Shock Protein Family A (Hsp70) Member 9 (HSPA9) and Member 8 (HSPA8). For HTR8/SVneo, altered proteins including prostaglandin F2α receptor regulatory protein (FPRP), RAB5A, RAB35, RAB5B, and RB11B, STAM1 and TSG101. These proteins are associated with the secretion and trafficking of EVs, which could explain in part, changes in the levels of circulating EVs in diabetic pregnancies. Further, we identified that proteins RAB11B, PDCD6IP, STAM, HSPA9, HSPA8, SDCBP, RAB5B, RAB5A, RAB7A and ERAP1 regulate EV release in response to high and low glucose when overexpressed in cells. Interestingly, immunohistochemistry analysis of RAB7A revealed distinct changes in placental tissues obtained from women with normal glucose tolerance (NGT, n = 6) and those with GDM (n = 6), influenced by diet or insulin treatment. High glucose regulation of proteins involved in intercellular dynamics and the trafficking of multivesicular bodies to the plasma membrane in placental cells is relevant in the context of GDM pregnancies.

Exploring Intercellular Dynamics: Ultra-Weak Biophoton Emission as a Novel Indicator of Altered Cell Functions and Disease in Oligospermia Mice.

Introduction: Biophoton emission, the spontaneous release of photons from living cells, has emerged as an attractive field of research in the study of biological systems. Scientists have recently discovered that changes in biophoton emission could serve as potential indicators of pathological conditions. This intriguing phenomenon suggests that cells might communicate and interact with each other through the exchange of these faint but significant light signals. Therefore, the present study introduces intercellular relationships with biophoton release to detect normal and abnormal cell functions to further achieve cellular interactions by focusing on cell and cell arrangement in disease conditions. Methods: Twenty male mice were assigned to control and busulfan groups. Five weeks after the injection of busulfan, the testis was removed, and then the stereological techniques and TUNEL assay were applied to estimate the histopathology of the testis tissue sections. Results: The findings revealed that the ultra-weak biophoton emission in the control group was significantly lower than in the busulfan group. The oligospermia mice model showed that it significantly changed the spatial arrangement of testicular cells and notably decreased the testis volume, length of seminiferous tubules, and the number of testicular cells. The results of the TUNEL assay showed that the percentage of apoptotic cells significantly increased in the busulfan group. Conclusion: The ultra-weak biophoton emission from testis tissue was reduced in oligospermia mice. As a result, the decline of ultra-weak biophoton can indicate a change in cell arrangement, a decrease in intercellular interaction, and eventually disease.

Mimicking acute airway tissue damage using femtosecond laser nanosurgery in airway organoids.

Airway organoids derived from adult murine epithelial cells represent a complex 3D in vitro system mimicking the airway epithelial tissue's native cell composition and physiological properties. In combination with a precise damage induction via femtosecond laser-based nanosurgery, this model might allow for the examination of intra- and intercellular dynamics in the course of repair processes with a high spatio-temporal resolution, which can hardly be reached using in vivo approaches. For characterization of the organoids' response to single or multiple-cell ablation, we first analyzed overall organoid survival and found that airway organoids were capable of efficiently repairing damage induced by femtosecond laser-based ablation of a single to ten cells within 24h. An EdU staining assay further revealed a steady proliferative potential of airway organoid cells. Especially in the case of ablation of five cells, proliferation was enhanced within the first 4h upon damage induction, whereas ablation of ten cells was followed by a slight decrease in proliferation within this time frame. Analyzing individual trajectories of single cells within airway organoids, we found an increased migratory behavior in cells within close proximity to the ablation site following the ablation of ten, but not five cells. Bulk RNA sequencing and subsequent enrichment analysis revealed the differential expression of sets of genes involved in the regulation of epithelial repair, distinct signaling pathway activities such as Notch signaling, as well as cell migration after laser-based ablation. Together, our findings demonstrate that organoid repair upon ablation of ten cells involves key processes by which native airway epithelial wound healing is regulated. This marks the herein presented in vitro damage model suitable to study repair processes following localized airway injury, thereby posing a novel approach to gain insights into the mechanisms driving epithelial repair on a single-cell level.

A transformable three-dimensional simulated solid tumor model reveals the widespread killing potential of natural killer cells with controlled lytic granule positioning

Abstract Cancer immunotherapy using engineered natural killer (NK) cells has shown great promise. However, the limited spatial complexity of current models hinders the study of NK cells in three-dimensional, solid tumor-like environments. Previously, we found that NK cells converge lytic granules before degranulation. It helps focus the secretion of cytotoxic cargos to the synaptic cleft, thus preventing the non-specific killing of bystander cells. We had speculated that bypassing this protective mechanism and forcing NK cells to degranulate non-directionally may promote broader destruction of cancer cells and eradicate tumor-resident non-triggering cells. To evaluate our hypothesis, we designed a novel strategy for creating complex cellular environments with controlled spatial distribution and intercellular dynamics. Using hydrogel-embedded cells as building blocks, we construct 3D models that arrange multiple cell populations within desired inner structures. Notably, these structures can undergo controlled transformations enabled by hydrogels comprised of thermo-responsive polymers. During this transformation, cells experienced rapid but mechanically gentle translocation to pre-designated positions, allowing us to control cell contacts actively. With these systems (which we have termed TheCOS for Thermal Collapse Of Strata), we observed that controlling NK cell lytic granule positioning at a lytic immune synapse was able to promote bystander cell death within a complex multicellular environment. Using quantitative imaging, we calculated an area of damage attributable to radial degranulation, which suggests a potential benefit to this as a strategy for improving cytotoxic cell therapy for solid tumors.

Single-objective selective plane illumination microscopy for multi-well organoid imaging.

Current preclinical in vitro and non-human animal models fail to adequately reproduce human physiology and disease processes. While recent efforts have focused on using microfluidics to help create stem cell-derived organoids as a way to better model 3D vascularization and improve the delivery of therapeutics, there remain challenges especially in terms of long-term imaging. To address this, we report here on the development of single-objective Selective Plane Illumination Microscopy (SoSPIM) for high-content imaging. This platform is capable of imaging samples in different mounting formats, ranging from tissue slices, to conventional culture dishes and and multi-well plates. To demonstrate this platform's capabilities, we imaged cerebral organoid slices and fixed organoids cultured in multi-well microfluidic devices. Our future work will focus on applying SoSPIM for long-term live organoid imaging using machine learning approaches for tracking and mapping intercellular dynamics and structures.

Abstract 2445: Orchestrated expression of the atypical Rho-GTPase RHOV mediates multicellular aggregate formation in ovarian cancer

Abstract Transcoelomic spread is a major mode of ovarian cancer (OVCA) metastasis, where cells disseminate into the peritoneal ascites fluid, evade anoikis, and form multi-cellular aggregates (MCAs) that mediate anchorage-independent (a-i) survival and invasion of the peritoneal organs. Isolation of MCAs from patient ascites correlates with poor patient survival and resistance to therapy. However, the mechanisms promoting anoikis evasion and OVCA MCA formation in a-i are not fully understood. Our previous data from patient ascites-derived cells, cell lines, and in vivo studies, demonstrate that OVCA cells upregulate the nutrient/stress sensor SIRT3 in response to a-i. We further illustrated that SIRT3 is necessary for the activation of the primary mitochondrial dismutase, SOD2, which in turn promotes the mitochondrial antioxidant defense to maintain optimal survival in a-i. To identify additional key molecular players important for a-i survival downstream of SIRT3, we employed RNA sequencing analysis to compare the transcriptome of SIRT3 knockdown and wildtype cells in both attached and a-i conditions using the serous adenocarcinoma cell line OVCA433. Analysis of early transcriptomic changes, observed within 2 hrs as well as 24 hrs of a-i, revealed that RHOV, a recently identified atypical member of the Rho GTPase family, is the top gene significantly upregulated in a-i. SIRT3 knock-down significantly decreases RHOV expression in 24hr a-i conditions, implicating RHOV as a potential downstream target of SIRT3 signaling in a-i. RHOV was reported to be overexpressed in lung cancer and found to correlate with poor patient outcome and resistance to therapy. RHOV expression also regulates intercellular adhesion dynamics during development. Our data show that genetic silencing of RHOV expression interferes with the ability of OVCA cells to form MCAs in a-i, suggesting that increased expression of RHOV in a-i conditions acts as an adaptive survival advantage mediating cell-cell adhesion and MCA formation. Current work is exploring how RHOV regulates E-cadherin in this context, by focusing on p21-stimulated serine/threonine protein kinase signaling. In addition, the mechanisms of SIRT3 dependent regulation are being explored by determining how changes in mitochondrial protein deacetylation contribute to RHOV transcription. This work highlights an expanding role of SIRT3 in mediating a-i survival of OVCA and explores the novel role of an understudied member of the Rho family of GTPases, RHOV during OVCA metastasis. Finally, given that the formation of MCAs has been correlated with resistance to therapy and increased rates of recurrence, we aim to test how MCA formation and targeting of key players of cell-cell adhesion in a-i, such as a RHOV, can be exploited for novel therapeutic strategies to prolong survival of OVCA patients diagnosed with metastatic disease. Citation Format: Amal T. Elhaw, Yeon Soo Kim, Zaineb Javed, Priscilla Tang, Weihua Pan, Nadine Hempel. Orchestrated expression of the atypical Rho-GTPase RHOV mediates multicellular aggregate formation in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2445.

A theoretical analysis of the scale separation in a model to predict solid tumour growth

Solid tumour growth depends on a host of factors which affect the cell life cycle and extracellular matrix vascularization that leads to a favourable environment. The whole solid tumour can either grow or wither in response to the action of the immune system and therapeutics. A personalised mathematical model of such behaviour must consider both the intra- and inter-cellular dynamics and the mechanics of the solid tumour and its microenvironment. However, such wide range of spatial and temporal scales can hardly be modelled in a single model, and require the so-called multiscale models, defined as orchestrations of single-scale component models, connected by relation models that transform the data for one scale to another. While multiscale models are becoming common, there is a well-established engineering approach to the definition of the scale separation, e.g., how the spatiotemporal continuum is split in the various component models. In most studies scale separation is defined as natural, linked to anatomical concepts such as organ, tissue, or cell; but these do not provide reliable definition of scales: for examples skeletal organs can be as large as 500 mm (femur), or as small as 3 mm (stapes). Here we apply a recently proposed scale-separation approach based on the actual experimental and computational limitations to a patient-specific model of the growth of neuroblastoma. The resulting multiscale model can be properly informed with the available experimental data and solved in a reasonable timeframe with the available computational resources.

Characterization of HIV-1 Infection in Microglia-Containing Human Cerebral Organoids.

The achievement of an HIV cure is dependent on the eradication or permanent silencing of HIV-latent viral reservoirs, including the understudied central nervous system (CNS) reservoir. This requires a deep understanding of the molecular mechanisms of HIV’s entry into the CNS, latency establishment, persistence, and reversal. Therefore, representative CNS culture models that reflect the intercellular dynamics and pathophysiology of the human brain are urgently needed in order to study the CNS viral reservoir and HIV-induced neuropathogenesis. In this study, we characterized a human cerebral organoid model in which microglia grow intrinsically as a CNS culture model to study HIV infection in the CNS. We demonstrated that both cerebral organoids and isolated organoid-derived microglia (oMG), infected with replication-competent HIVbal reporter viruses, support productive HIV infection via the CCR5 co-receptor. Productive HIV infection was only observed in microglial cells. Fluorescence analysis revealed microglia as the only HIV target cell. Susceptibility to HIV infection was dependent on the co-expression of microglia-specific markers and the CD4 and CCR5 HIV receptors. Altogether, this model will be a valuable tool within the HIV research community to study HIV–CNS interactions, the underlying mechanisms of HIV-associated neurological disorders (HAND), and the efficacy of new therapeutic and curative strategies on the CNS viral reservoir.

Magnetophoretic Micro‐Distributor for Controlled Clustering of Cells (Adv. Sci. 6/2022)

Magnetophoretic Micro-Distributor In article number 2103579, Byeonghwa Lim, CheolGi Kim, and co-workers propose a magnetophoretic device which consists of a trampoline micromagnet and active elements which enable the control of individual cell-based on physical variables for the automatic separation or selective clustering for intercellular communications. This technology could be a milestone for a better understanding of the intercellular dynamics between clustered cells at the single-cell level for future cell-on-chip applications.

High-Frequency Head Impact Disrupts Hippocampal Neural Ensemble Dynamics.

We have recently shown that the cognitive impairments in a mouse model of high-frequency head impact (HFHI) are caused by chronic changes to synaptic physiology. To better understand these synaptic changes occurring after repeat head impact, we used Thy1-GcCAMP6f mice to study intracellular and intercellular calcium dynamics and neuronal ensembles in HFHI mice. We performed simultaneous calcium imaging and local field potential (LFP) recordings of the CA1 field during an early-LTP paradigm in acute hippocampal slice preparations 24 h post-impact. As previously reported, HFHI causes a decrease in early-LTP in the absence of any shift in the input-output curve. Calcium analytics revealed that HFHI hippocampal slices have similar numbers of active ROIs, however, the number of calcium transients per ROI was significantly increased in HFHI slices. Ensembles consist of coordinated activity between groups of active ROIs. We exposed the CA1 ensemble to Schaffer-collateral stimulation in an abbreviated LTP paradigm and observed novel coordinated patterns of post stimulus calcium ensemble activity. HFHI ensembles displayed qualitatively similar patterns of post-stimulus ensemble activity to shams but showed significant changes in quantitative ensemble inactivation and reactivation. Previous in vivo and in vitro reports have shown that ensemble activity frequently occurs through a similar set of ROIs firing in a repeating fashion. HFHI slices showed a decrease in such coordinated firing patterns during post stimulus ensemble activity. The present study shows that HFHI alters synaptic activity and disrupts neuronal organization of the ensemble, providing further evidence of physiological synaptic adaptation occurring in the brain after a high frequency of non-pathological head impacts.

Magnetophoretic Micro-Distributor for Controlled Clustering of Cells.

Cell clustering techniques are important to produce artificial cell clusters for in vitro models of intercellular mechanisms at the single‐cell level. The analyses considering physical variables such as the shape and size of cells have been very limited. In addition, the precise manipulation of cells and control of the physical variables are still challenging. In this paper, a magnetophoretic device consisting of a trampoline micromagnet and active elements that enable the control of individual selective jumping motion and positioning of a micro‐object is proposed. Based on a numerical simulation under various conditions, automatic separation or selective clustering of micro‐objects according to their sizes is performed by parallel control and programmable manipulation. This method provides efficient control of the physical variables of cells and grouping of cells with the desired size and number, which can be a milestone for a better understanding of the intercellular dynamics between clustered cells at the single‐cell level for future cell‐on‐chip applications.

Next-Generation Human Cerebral Organoids as Powerful Tools To Advance NeuroHIV Research.

ABSTRACTLong-term effective use of antiretroviral therapy (ART) among people with HIV (PWH) has significantly reduced the burden of disease, yet a cure for HIV has not been universally achieved, likely due to the persistence of an HIV reservoir. The central nervous system (CNS) is an understudied HIV sanctuary. Importantly, due to viral persistence in the brain, cognitive disturbances persist to various degrees at high rates in PWH despite suppressive ART. Given the complexity and accessibility of the CNS compartment and that it is a physiologically and anatomically unique immune site, human studies to reveal molecular mechanisms of viral entry, reservoir establishment, and the cellular and structural interactions leading to viral persistence and brain injury to advance a cure and either prevent or limit cognitive impairments in PWH remain challenging. Recent advances in human brain organoids show that they can mimic the intercellular dynamics of the human brain and may recapitulate many of the events involved in HIV infection of the brain (neuroHIV). Human brain organoids can be produced, spontaneously or with addition of growth factors and at immature or mature states, and have become stronger models to study neurovirulent viral infections of the CNS. While organoids provide opportunities to study neuroHIV, obstacles such as the need to incorporate microglia need to be overcome to fully utilize this model. Here, we review the current achievements in brain organoid biology and their relevance to neuroHIV research efforts.

A novel tumor-immune microenvironment (TIME)-on-Chip mimics three dimensional neutrophil-tumor dynamics and neutrophil extracellular traps (NETs)-mediated collective tumor invasion

Neutrophils are the most abundant type of leukocytes in the blood, traditionally regarded as the first immune responders to infections and inflammations. In the context of tumors, neutrophils have been shown to possess both tumor-promoting and tumor-limiting properties. A better understanding of the inter-cellular dynamics between the neutrophils and aggregated tumors could possibly shed light on the different modalities of neutrophil involvement in tumor progression. To study in-vitro the interactional dynamics of neutrophils and growing tumor aggregates, in this work, we engineered a novel, microfluidics-integrated, three-dimensional (3D) tumor-immune microenvironment (TIME)-on-Chip device, and we investigated the effect of neutrophils on the inception of collective 3D invasion of ovarian tumor cells. Herein, tumor spheroids generated and cultured on hydrogel based multi-microwell plates, and embedded within collagen matrix of defined thickness, were magnetically hybrid-integrated with a 3D bioprinting enabled microfluidic system fabricated on a porous membrane and carrying neutrophils. This setting recreated a typical TIME in-vitro to model dynamic neutrophil migration and 3D tumor invasion. Using this device, we observed that neutrophils respond to the growing tumor spheroids through both chemotaxis and generation of neutrophil extracellular traps (NETs). The formation of NETs stimulated the reciprocation of tumor cells from their aggregated state to collectively invade into the surrounding collagen matrix, in a manner more significant compared to their response to known tumor-derived stimulants such as transforming growth factor and Interleukin- 8. This effect was reversed by drug-induced inhibition of NETs formation, suggesting that induction of NETs by cancer cells could be a pro-migratory tumor behavior. Further, we additionally report a previously unidentified, location-dictated mechanism of NETosis, in which NETs formation within the stromal extracellular collagen matrix around the spheroids, and not tumor-contacted NETs, is important for the induction of collective invasion of the ovarian tumor cells, thus providing a rationale for new anti-tumor therapeutics research.

Fibronectin-induced ductal formation in salivary gland self-organization model.

Recent advances in tissue regeneration approaches including 3D organoids, were based on various 3D organogenesis models. However, 3D models are generally technique-sensitive and time-consuming. Thus, we utilized an existing model of submandibular salivary gland (SMG) to modify a simple and highly reproducible in vitro 3D culture model of primary SMG cells self-organization into a well-developed cell spheroid inside Matrigel substrate. We used this model to observe the collective multicellular behavior during spheroid formation. Further, we applied various quantitative approaches including real-time live imaging and immune histochemical image analysis to dissect the cellular dynamics during tissue patterning. On a time-scale of hours, we observed marked size and shape transformations in the developed 3D spheroid which resulted in a spatially-controlled growth differential from the canter to the periphery of the formed aggregates. Moreover, we investigated the effect of fibronectin (FN) on SMG cells self-organization using our simplified culture model. Interestingly, we discovered a novel role of FN in inducing duct-like elongation during initial stages of SMG bud formation. This in vitro model provides an excellent tool for analyzing the intercellular dynamics during early SMG tissue development as well as revealing a novel role of FN in SMG ductal expansion.

Cell-cell adhesion interface: orthogonal and parallel forces from contraction, protrusion, and retraction.

The epithelial lateral membrane plays a central role in the integration of intercellular signals and, by doing so, is a principal determinant in the emerging properties of epithelial tissues. Mechanical force, when applied to the lateral cell–cell interface, can modulate the strength of adhesion and influence intercellular dynamics. Yet the relationship between mechanical force and epithelial cell behavior is complex and not completely understood. This commentary aims to provide an investigative look at the usage of cellular forces at the epithelial cell–cell adhesion interface.