Migratory Cells Research Articles

CD19 CAR-expressing iPSC-derived NK cells effectively enhance migration and cytotoxicity into glioblastoma by targeting to the pericytes in tumor microenvironment

In cancer immunotherapy, chimeric antigen receptors (CARs) targeting specific antigens have become a powerful tool for cell-based therapy. CAR-natural killer (NK) cells offer selective anticancer lysis with reduced off-tumor toxicity compared to CAR-T cells, which is beneficial in the heterogeneous milieu of solid tumors. In the tumor microenvironment (TME) of glioblastoma (GBM), pericytes not only support tumor growth but also contribute to immune evasion, underscoring their potential as therapeutic targets in GBM treatment. Given this context, our study aimed to target the GBM TME, with a special focus on pericytes expressing CD19, to evaluate the potential effectiveness of CD19 CAR-iNK cells against GBM. We performed CD19 CAR transduction in induced pluripotent stem cell-derived NK (iNK) cells. To determine whether CD19 CAR targets the TME pericytes in GBM, we developed GBM-blood vessel assembloids (GBVA) by fusing GBM spheroids with blood vessel organoids. When co-cultured with GBVA, CD19 CAR-iNK cells migrated towards the pericytes surrounding the GBM. Using a microfluidic chip, we demonstrated CD19 CAR-iNK cells’ targeted action and cytotoxic effects in a perfusion-like environment. GBVA xenografts recapitulated the TME including human CD19-positive pericytes, thereby enabling the application of an in vivo model for validating the efficacy of CD19 CAR-iNK cells against GBM. Compared to GBM spheroids, the presence of pericytes significantly enhanced CD19 CAR-iNK cell migration towards GBM and reduced proliferation. These results underline the efficacy of CD19 CAR-iNK cells in targeting pericytes within the GBM TME, suggesting their potential therapeutic value for GBM treatment.

The Long Noncoding RNA Gall Bladder Cancer-Associated Suppressor of Pyruvate Carboxylase Inhibits the Proliferation, Migration, and Invasion of Colorectal Cancer Cells and Induces Their Apoptosis.

This study aimed to determine the role of the long noncoding RNA (lncRNA) gall bladder cancer-associated suppressor of pyruvate carboxylase (SOD2-1) in the progression of colorectal cancer (CRC). A total of 23 pairs of specimens, including CRC tissues and adjacent normal tissues, were collected, and the expression of lncRNA SOD2-1 (lnc-SOD2-1) was measured. lnc-SOD2-1 function was examined using HCT15 and HCT116 cells. A lnc-SOD2-1 overexpression vector was designed and transfected into both cell lines. MTS and colony formation assays were used to determine cell viability. Flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assays were performed to measure apoptosis. Cell migration and invasion were evaluated using the Transwell assay. Migration and invasion markers were validated using quantitative reverse transcription-polymerase chain reaction and western blot analysis. The results indicated that the expression of lnc-SOD2-1 was downregulated in CRC tissues. lnc-SOD2-1 overexpression evidently decreased cell viability and led to the formation of fewer cell colonies. lnc-SOD2-1 overexpression induced ~ twofold higher apoptosis than the control group. lnc-SOD2-1 overexpression reduced the proportion of migratory and invasive cells to 50% and 75% of the control group, respectively. lnc-SOD2-1 overexpression significantly decreased the expression of matrix metalloproteinase-2 and -9. In conclusion, lnc-SOD2-1 may act as a tumor suppressor that inhibits the proliferation, migration, and invasion of CRC cells and induces their apoptosis.

Dsg2 ectodomain organization increases throughout desmosome assembly

ABSTRACT Desmosomes are intercellular junctions that regulate mechanical integrity in epithelia and cardiac muscle. Dynamic desmosome remodeling is essential for wound healing and development, yet the mechanisms governing junction assembly remain elusive. While we and others have shown that cadherin ectodomains are highly organized, how this ordered architecture emerges during assembly is unknown. Using fluorescence polarization microscopy, we show that desmoglein 2 (Dsg2) ectodomain order gradually increases during 8 h of assembly, coinciding with increasing adhesive strength. In a scratch wound assay, we observed a similar increase in order in desmosomes assembling at the leading edge of migratory cells. Together, our findings indicate that cadherin organization is a hallmark of desmosome maturity and may play a role in conferring adhesive strength.

Sublingual allergen immunotherapy prevents house dust mite inhalant type 2 immunity through dendritic cell-mediated induction of Foxp3+ regulatory T cells

Sublingual allergen immunotherapy (SLIT) is an emerging treatment option for allergic asthma, and a potential disease modifying strategy for asthma prevention. The key cellular events leading to such long term tolerance remains to be fully elucidated. We administered prophylactic SLIT in a mouse model of house dust mite (HDM) driven allergic asthma. HDM extract was sublingually administered over 3 weeks followed by intratracheal sensitization and intranasal challenges with HDM. Prophylactic SLIT prevented allergic airway inflammation and hyperreactivity with a low lab-to-lab variation. The HDM specific Th2 (CD4 T helper) response was shifted by SLIT towards a regulatory and Th17 response in lung and mediastinal lymph node (MLN). By using Der p 1 specific CD4+ T cells (1-DER), we found that SLIT blocked 1-DER T cell recruitment to the MLN and dampened IL-4 secretion following intratracheal HDM sensitization. Sublingually administered Der p 1 protein activated 1-DER T cells in the cervical lymph node (CLN) via CCR7+ migratory dendritic cells (DC). DCs migrating from the oral submucosa to the CLN after SLIT induced Foxp3+ regulatory T cells. When mice were sensitized with HDM, prior prophylactic SLIT increased Der p 1 specific Tregs and lowered Th2 recruitment in the lung. By using Foxp3-DTR mice, Tregs were found to contribute to the immunoregulatory prophylactic effect of SLIT on type 2 immunity. These findings in a mouse model suggest that DC-mediated functional Treg induction in oral mucosa draining LNs is one of the driving mechanisms behind the disease modifying effect of prophylactic SLIT.

Cell migration: Collective cell migration is intrinsically stressful

Collective cell migration is a key cellular process in development and disease. A new study reports that ER stress is induced during collective cell migration and an intrinsic mechanism prevents migratory cells from over-reacting to ER stress.

MiR-522-3p Attenuates Cardiac Recovery by Targeting FOXP1 to Suppress Angiogenesis.

Angiogenesis is crucial for blood supply reconstitution after myocardial infarction in patients with acute coronary syndrome (ACS). MicroRNAs are recognized as important epigenetic regulators of endothelial angiogenesis. The purpose of this study is to determine the roles of miR-522-3p in angiogenesis after myocardial infarction. The expression levels of miR-522-3p in rats' plasma and in the upper part of the ligation of the heart tissues at 28 days after myocardial infarction were significantly higher than those of the sham group. miR-522-3p mimics inhibited cell proliferations, migrations, and tube formations under hypoxic conditions in HUVECs (human umbilical vein endothelial cells), whereas miR-522-3p inhibitors did the opposite. Furthermore, studies have indicated that the inhibition of miR-522-3p by antagomir infusion promoted angiogenesis and accelerated the recovery of cardiac functions in rats with myocardial infarction.Data analysis and experimental results revealed that FOXP1 (Forkhead-box protein P1) was the target gene of miR-522-3p. Our study explored the mechanism of cardiac angiogenesis after myocardial infarction and provided a potential therapeutic approach for the treatment of ischemic heart disease in the future.

Glioblastoma U-87 cell electrotaxis is hindered by doxycycline with a concomitant reduction in the matrix metallopeptidase-9 expression

Electric fields (EF) play an essential role in cancer cell migration. Numerous cancer cell types exhibit electrotaxis under direct current electric fields (dcEF) of physiological electric field strength (EFs). This study investigated the effects of doxycycline on the electrotactic responses of U87 cells. After EF stimulation, U87 cells migrated toward the cathode, whereas doxycycline-treated U87 cells exhibited enhanced cell mobility but hindered cathodal migration. We further investigated the expression of the metastasis-correlated proteins matrix metallopeptidase-2 (MMP-2) and MMP-9 in U87 cells. The levels of MMP-2 in the cells were not altered under EF or doxycycline stimulation. In contrast, the EF stimulation greatly enhanced the levels of MMP-9 and then repressed in doxycycline-cotreated cells, accompanied by reduced cathodal migration. Our results demonstrated that an antibiotic at a non-toxic concentration could suppress the enhanced cell migration accelerated by EF of physiological strength. This finding may be applied as an anti-metastatic treatment for cancers.

Abstract 272: Novel role for eIF4E in homing of acute myeloid leukemia cells

Abstract Acute myeloid leukemia (AML) remains with dismal outcomes, mostly attributed to the inability of therapies to eliminate leukemia stem cells (LSCs) leading to relapse. LSCs home to the bone marrow (BM) niche where they maintain their quiescence state once in contact with mesenchymal stromal cells (MSCs). Therefore, understanding what controls AML-niche interactions could lead to therapeutic approaches that can prevent relapse. First, we implemented a novel 3D in vitro system to mimic human AML-niche interactions. This 3D structure consists of a spheroid formed with BM-MSCs (HS5 cell line), recreating a hypoxic microenvironment. When AML cells (MM6 cell line) are added to the spheroid in a 1:1 proportion, AML cells migrate to the spheroid. After 24 hours, 0.18% of the AML cells home/colonize the spheroid (n=8). Next, we assessed the cell cycle status of AML cells in co-culture with the spheroid and compare to those in liquid culture or placed on a MSC monolayer (2D). We found that cells upon contact with MSCs increased their quiescence, 18.63% G0 in 2D and significantly higher in 3D conditions (25.03%; p=0.0048). It is also important to point out that colonizing cells have a lower proliferate capacity than non-colonizing cells after 7 days of co-culture (p=0.0286). This quiescence feature suggests a stem-like behavior of cells homing to the spheroid. Eukaryotic translation initiation factor 4E (eIF4E) has been found altered in AML. While the capacity of eIF4E to increase cell migration and growth has been shown in solid cancers, the mechanism of eIF4E to accelerate AML progression remains unknown. Thus, we hypothesized that eIF4E may play a role in homing of AML cells to the niche. To this end, we used CRISPR to deplete eIF4E in MM6 cells and found that CRISPR 4E cells have significantly decreased homing capacity to the spheroid compared with control cells (p=0.0065). Importantly, we found a lower percentage of CRISPR 4E cells underwent quiescence after co-culture with MSCs (2D or 3D) (p<0.0001). We corroborated observations using xenograft mice. After 20 hours of intravenous injection, we observed a higher percentage of AML cells in the blood of the mice injected with CRISPR 4E cells compared with control cells (p=0.0286), suggesting that CRISPR 4E cells are not able to effectively exit from peripheral blood to home to their niche. Altogether, we stablished a novel in vitro 3D model that mimics stem cell features of the AML-BM niche. We demonstrated that the depletion of eIF4E in AML cells decreases their homing and prevents them from acquiring niche-induced quiescence. Citation Format: Leandro M. Martinez, Eloisi Caldas Lopes, Mayumi Sugita, Jadwiga Gasiorek, Kata Alilovic, Katherine L. Borden, Monica L. Guzman. Novel role for eIF4E in homing of acute myeloid leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 272.

Abstract 1668: Association of stem cell-like phenotype with isoform specific functions of AKTs in hepatocytes

Abstract Introduction: Liver cancer is the sixth most common cancer and the third leading cause of cancer-related deaths worldwide. It is associated with abnormal activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which is implicated in regulation of cancer stem cells (CSCs). The present study investigated the effects of AKT loss on stem cell-like phenotype in immortalized mouse hepatocytes. Methods: Immortalized hepatocytes were established from mice with Akt1 (Akt1−/−; Akt1-null) or Akt2 (Akt2−/−; Akt2-null) whole-body deletion. Cell morphology was assessed via microscopy. Cell proliferation was evaluated with hemacytometer count and MTT assay. Cell migration towards a chemoattractant was assessed via transwell assay. Cell viability was evaluated in response to chemotherapy treatment. The ability of cells to Results: When compared to WT cells, which exhibited cuboidal epithelial-like features, Akt1-null and Akt2-null cells demonstrated elongated mesenchymal-like morphology. Loss of either isoform was associated with a significantly decreased cell growth (p<0.05). Akt1-null cells migrated faster towards a chemoattractant in the transwell assay, were significantly more resistant to sorafenib, and formed a significantly higher number of spheres in the sphere formation assay (p<0.01). Proteomic analysis revealed that both Akt1-null and Akt2-null cells exhibited increased expression of CSC marker CD44, along with other proteins associated with liver cancer, including PDGFRA, CDH2, MMP14, and COX2 (p<0.05). Additionally, Akt1-null cells exhibited elevated levels of ANXA1 and ANXA3 (p<0.01). Conclusions: Loss of both AKT isoforms resulted in decreased cell proliferation and elevated expression of proteins with a previously reported role in liver cancer invasion, migration, and epithelial-mesenchymal transition. AKT1 loss correlated with an enhanced stem cell-like phenotype, including increased migration towards a chemoattractant, higher resistance to chemotherapy, and enhanced ability to form spheres. These results contribute to the understanding of the role of AKT isoforms in regulating stemness in liver cancer and may serve as a basis for investigation into isoform specific inhibitors. Further investigation into cellular mechanisms governing these phenotypes is required. Citation Format: Ielyzaveta Slarve, Yushan Wang, Mario Alba, Lina He, Bangyan Stiles. Association of stem cell-like phenotype with isoform specific functions of AKTs in hepatocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1668.

Abstract 365: Proteomic analysis of cytoplasmic lipid droplets and whole cell lysates reveal ferroptosis regulation by fatty acid synthase-derived lipid droplets in metastatic breast cancer

Abstract Breast cancer remains a serious public health concern, where metastasis accounts for the majority of patient mortality. Lipid accumulation in metastases is associated with reduced treatment response and poorer patient outcomes, although the mechanisms linking lipid accumulation and metastasis are not clear. We previously demonstrated that metastatic MCF10CA1a breast cancer cells have higher de novo lipogenesis and cytoplasmic lipid droplets (CLDs) compared to non-metastatic MCF10A-ras cells. Additionally, fatty acid synthase (FASN)-derived TAG stores sustained key metastatic processes in the MCF10CA1a cells. The proteome of CLDs is diverse and plastic, reflecting differing CLD functions; therefore, we sought to identify proteins differentially abundant on CLDs from vehicle-treated and FASN-inhibited MCF10CA1a breast cancer cells. We performed untargeted, global proteomics analysis on whole cell lysates (WCLs) and CLDs from vehicle-treated and FASN-inhibited (TVB-3166) MCF10CA1a cells to determine protein pathways differentially enriched on CLDs. Together, 2,416 and 3,539 proteins were identified within CLD fractions and WCLs, respectively. Of the proteins identified in CLD fractions, ~30% (717) were commonly identified within both conditions. Interestingly, 1,571 proteins were unique to the CLD fraction from vehicle-treated cells, whereas only 19 proteins were unique to the CLD fraction from FASN-inhibited cells. Proteins unique to CLDs from vehicle-treated cells include those known to reduce cancer cell survival and progression, such as proteins involved in cadherin-binding and cell cycle regulation. Proteins known to reduce cancer progression that associate with CLDs may prevent them from functioning at their normal site of action. Interestingly, an inhibitor of ferroptosis, glutathione peroxidase 4 (GPX4), was only detected in the vehicle-treated CLD and WCL samples, whereas proteins that promote ferroptosis were more abundant on CLDs of the more migratory cells, although their levels between WCLs were similar. Taken together, these data suggested that the machinery for ferroptosis may be mislocalized in TAG-rich cells, thereby protecting them from ferroptosis. Indeed, FASN-inhibited cells had nearly 30% more intracellular iron compared to the vehicle-treated cells, an important prerequisite for ferroptosis, thus indicating higher levels of ferroptotic stress. Functionally, the vehicle-treated cells were 25% more readily rescued by N-acetylcysteine from ferroptosis induced by erastin than were FASN-inhibited cells, despite comparable sensitivity to erastin. These preliminary findings suggest that FASN-derived CLDs in metastatic cells may aid in protecting against ferroptosis to promote breast cancer progression. Citation Format: Chaylen Andolino, Kimberly K. Buhman, Michael F. Coleman, Stephen D. Hursting, Marjorie Layosa, Michael K. Wendt, Dorothy Teegarden. Proteomic analysis of cytoplasmic lipid droplets and whole cell lysates reveal ferroptosis regulation by fatty acid synthase-derived lipid droplets in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 365.

Abstract 2852: Novel combination therapy with a KAT6A/B inhibitor together with retinoids induces irreversible differentiation of neuroblastoma cells

Abstract Neuroblastoma is a pediatric tumor of the peripheral sympathetic nervous system derived from migratory neural crest cells that have committed to become sympathetic neuroblasts, but their definitive differentiation is blocked. Neuroblastoma cell identity depends on the adrenergic core regulatory circuit (CRC) of transcription factors that collaborate with MYCN to drive cell proliferation and the oncogenic gene expression program. Retinoic acid is used as differentiation therapy for patients with high-risk neuroblastoma, with the goal to block proliferation and drive sympathetic neuronal differentiation. We recently showed that retinoic acid re-organizes the enhancer landscape of neuroblastoma and promotes a new retino-sympathetic cell state characterized by MYCN downregulation, proliferative arrest, and sympathetic neuronal differentiation. However, the effects of retinoic acid on epigenetic rewiring of the adrenergic CRC and suppression of MYCN expression are reversible in vitro and in vivo, leading to tumor re-growth when the retinoic acid is withdrawn. Therefore, we sought to determine if retinoid-induced differentiation can be forced to progress to irreversible neuronal maturation by the addition of inhibitors of epigenetic modifying enzymes. Here, we conducted a drug screen of epigenetic modifying drugs alone and in combination with retinoic acid to determine their effects on neuroblastoma cell growth and differentiation. In this screen, we found that an inhibitor of the histone H3K23 acetyltransferases KAT6A/B, PF-9363, synergistically inhibits neuroblastoma cell growth in combination with retinoic acid. Moreover, retinoid-resistant neuroblastoma cell lines can be sensitized to retinoic acid when combined with a KAT6 inhibitor. Importantly, this combination treatment renders the differentiated cell state irreversible, such that it is maintained after retinoic acid is withdrawn, with continued suppression of the adrenergic CRC and MYCN expression. In conclusion, the retino-sympathetic differentiated cell state induced by retinoic acid in neuroblastoma becomes irreversible when the cells are also treated with an inhibitor of the KAT6A/B histone H3K23 acetyltransferases, implicating the essential role of these enzymes in restoring the proliferative immature progenitor phenotype in adrenergic neuroblastoma cells. Citation Format: Nina Weichert-Leahey, Alla Berezovskaya, Mark Zimmerman, Brian J. Abraham, Adam W. Durbin, A Thomas Look. Novel combination therapy with a KAT6A/B inhibitor together with retinoids induces irreversible differentiation of neuroblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2852.

Abstract 1504: Tumor microenvironment-mediated regulation of early dissemination and colonization in breast cancer via BAZ2A-containing remodeling complexes

Abstract The molecular mechanisms governing invasion, dissemination, extravasation, and early metastatic colonization remain incompletely understood. In the BALB-neuT mouse model, we found that most metastases originate from disseminated cancer cells (DCCs) that left the primary site early in tumorigenesis, i.e. when lesions are still small. Consequently, we investigated whether cellular density and the tumor microenvironment synergize to generate the plasticity needed during metastasis. RNA-sequencing analyses of murine breast cancer cells in low cellular density (LD) and high cellular density (HD) conditions revealed distinct profiles of differentially expressed genes. Notably, we identified the enrichment of ribosomal RNA (rRNA) and chromatin remodeling pathways in LD conditions. Interestingly, once these cells were treated with Wnt4 and Rankl, factors secreted by the tumor microenvironment, the expression profile of genes involved in proliferation, migration and stemness as well as epigenetic-related factors changed drastically. Specifically, cells in LD conditions treated with Wnt4 and Rankl displayed induced migration and stemness phenotypes, whereas under HD conditions, these signals induced proliferation. Among the chromatin-remodeling pathways, we found that BAZ2A along with the rRNA regulating network were primarily upregulated in migratory cancer cells. Immunostaining of primary and metastatic lesions in murine and human breast cancer samples showed an upregulation of BAZ2A in early/microlesions compared to more advanced and proliferative macroscopic lesions. Taken together, these results indicate that cell density, tumor microenvironmental factors and BAZ2A-containing chromatin regulating complexes play pivotal roles in regulating cancer cell plasticity in breast cancer. Additional sequencing and functional analyses will be performed to dissect the specific components of this mechanism and evaluate their impact on the in vivo colonization of breast cancer models. Citation Format: Miriam Kokal-Ribaudo, Nora Jahnen, Saba Sameri, Fulvia Ferrazzi, Renato Liguori, Christoph A. Klein, Gernot Längst, Hedayatollah Hosseini. Tumor microenvironment-mediated regulation of early dissemination and colonization in breast cancer via BAZ2A-containing remodeling complexes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1504.

Abstract 6802: CXCR4 transgene improves in vivo migration and efficacy of engineered iPSC-derived natural killer cells

Abstract Chimeric antigen receptor (CAR)-engineered induced pluripotent stem cell (iPSC)-derived natural killer (iNK) cells have shown a favorable safety profile and early evidence of clinical efficacy in the treatment of patients with certain hematologic malignancies. CAR-iNK cells retain phenotypic and functional similarities to primary natural killer cells, with additional capabilities for recognition of tumors. For the treatment of cancers that are located in the bone marrow, it is critical that iNK cells are able to migrate efficiently to relevant sites of disease. CXCR4 (C-X-C chemokine receptor type 4) is a chemokine receptor specific for stromal-derived-factor-1 (SDF-1), and mediates the migration of hematopoietic cells to the bone marrow upon binding SDF-1. Here we describe novel engineering of iPSCs resulting in efficient migration and efficacy of iNK cells. iPSC were sequentially engineered at defined loci with Crispr/MAD7/HDR to incorporate Allo-EvasionTM edits, a CAR targeting antigens expressed on different hematological malignancies, constitutive interleukin-15 (IL-15) cytokine support, with and without wild-type CXCR4. iPSCs were then differentiated into natural killer cells using a proprietary differentiation process. Cytolysis of a panel of target cells by iNK was evaluated using an Incucyte-based killing assay. Anti-tumor efficacy was tested in disseminated xenograft models of hematological malignancies following intravenous injection of engineered iNK cells. Additionally, in vivo migration was evaluated using immunohistochemistry (IHC) and flow cytometry (FACS) of blood, bone marrow, and other organs. Engineering with CXCR4 resulted in a bulk population of iNK with a heterogenous expression of CXCR4. No significant impact of CXCR4 engineering was detected on iPSC differentiation, phenotype, or iNK yield. iNKCXCR4+ and iNKCXCR4− cells had equivalent target tumor cell cytolysis in vitro, indicating the CXCR4 transgene does not affect killing potential. In vivo, iNKCXCR4+ cells migrated to the bone marrow of both tumor-bearing and non-tumor-bearing animals, and the presence of these cells was enhanced in tumor-bearing mice, indicating CXCR4-mediated migration and possibly CAR-mediated expansion and/or retention of iNK. Migration to the bone marrow was not observed with iNKCXCR4− cells. As a result, iNKCXCR4+ cells significantly impacted anti-tumor efficacy, effectively eliminating bone disease. We have demonstrated the CAR-specific killing and enhanced iNK migration and subsequent tumor killing in the bone marrow, with the addition of a CXCR4 transgene in iPSC-derived NK cells. The addition of the CXCR4 transgene in iPSC-derived effector cells may allow for a higher therapeutic response in axial-skeletal located tumors. Citation Format: Hillary J. Millar, David Walker, Liam R. Campion, Ciara Budd, Aarti Kolluri, Harsh Sharma, Buddha Gurung, Jill M. Carton, Daniel J. Perry, Nicholas Alexander, Hyam Levitsky, Michael Naso, Barry A. Morse. CXCR4 transgene improves in vivo migration and efficacy of engineered iPSC-derived natural killer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6802.

CD56/NCAM mediates cell migration of human NK cells by promoting integrin-mediated adhesion turnover.

Natural killer (NK) cells patrol tissue to mediate lysis of virally infected and tumorigenic cells. Human NK cells are typically identified by their expression of neural cell adhesion molecule (NCAM, CD56), yet, despite its ubiquitous expression on NK cells, CD56 remains a poorly understood protein on immune cells. CD56 has been previously demonstrated to play roles in NK cell cytotoxic function and cell migration. Specifically, CD56-deficient NK cells have impaired cell migration on stromal cells and CD56 is localized to the uropod of NK cells migrating on stroma. Here, we show that CD56 is required for NK cell migration on ICAM-1 and is required for the establishment of persistent cell polarity and unidirectional actin flow. The intracellular domain of CD56 (NCAM-140) is required for its function and the loss of CD56 leads to enlarged actin foci and sequestration of phosphorylated Pyk2 accompanied by increased size and frequency of activated LFA-1 clusters. Together, these data identify a role for CD56 in regulating human NK cell migration through modulation of actin dynamics and integrin turnover. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].

The genome of the colonial hydroid Hydractinia reveals that their stem cells use a toolkit of evolutionarily shared genes with all animals.

Hydractinia is a colonial marine hydroid that shows remarkable biological properties, including the capacity to regenerate its entire body throughout its lifetime, a process made possible by its adult migratory stem cells, known as i-cells. Here, we provide an in-depth characterization of the genomic structure and gene content of two Hydractinia species, Hydractinia symbiolongicarpus and Hydractinia echinata, placing them in a comparative evolutionary framework with other cnidarian genomes. We also generated and annotated a single-cell transcriptomic atlas for adult male H. symbiolongicarpus and identified cell-type markers for all major cell types, including key i-cell markers. Orthology analyses based on the markers revealed that Hydractinia's i-cells are highly enriched in genes that are widely shared amongst animals, a striking finding given that Hydractinia has a higher proportion of phylum-specific genes than any of the other 41 animals in our orthology analysis. These results indicate that Hydractinia's stem cells and early progenitor cells may use a toolkit shared with all animals, making it a promising model organism for future exploration of stem cell biology and regenerative medicine. The genomic and transcriptomic resources for Hydractinia presented here will enable further studies of their regenerative capacity, colonial morphology, and ability to distinguish self from nonself.

Roles of Mechanosensitive Channel Piezo1 in Wound Healing and Scar Formation.

The ability to heal one's wounds is perhaps one of the most fundamental and critical of physiologic processes. This coordinated and closely regulated sequential biological process involves a variety of migratory and resident cells. The activation, modulation, balance, and control of these functions depend upon soluble mediators that activate cells and modulate their diverse functions. Recent advances have identified mechanotransduction as functionally integral in many different cell types and physiologic processes. The mechanically sensitive ion channel Pieoz1 is expressed on platelets, neutrophils, macrophages, endothelial cells, keratinocytes, and fibroblasts, all of which are principally involved in wound healing. On a cellular level, there have been great advances in our understanding of the functional role of Piezo1 mechanotransduction in cutaneous wounding. The blocking of Piezo1 has recently been shown to reduce scarring in vivo and yet, thus far, a comprehensive understanding of the roles that Piezo1 plays in in vivo wound healing remains lacking. Recognizing the ever-present and critical importance of optimal and reparative wound healing, and with the availability of new physical mechanomodulating devices, the time is ripe for gaining deeper insights into optimizing wound healing. In this review, we describe the current knowledge of Piezo1 related to wound healing.

Mitochondrial metabolism sustains CD8+ T cell migration for an efficient infiltration into solid tumors

The ability of CD8+ T cells to infiltrate solid tumors and reach cancer cells is associated with improved patient survival and responses to immunotherapy. Thus, identifying the factors controlling T cell migration in tumors is critical, so that strategies to intervene on these targets can be developed. Although interstitial motility is a highly energy-demanding process, the metabolic requirements of CD8+ T cells migrating in a 3D environment remain unclear. Here, we demonstrate that the tricarboxylic acid (TCA) cycle is the main metabolic pathway sustaining human CD8+ T cell motility in 3D collagen gels and tumor slices while glycolysis plays a more minor role. Using pharmacological and genetic approaches, we report that CD8+ T cell migration depends on the mitochondrial oxidation of glucose and glutamine, but not fatty acids, and both ATP and ROS produced by mitochondria are required for T cells to migrate. Pharmacological interventions to increase mitochondrial activity improve CD8+ T cell intratumoral migration and CAR T cell recruitment into tumor islets leading to better control of tumor growth in human xenograft models. Our study highlights the rationale of targeting mitochondrial metabolism to enhance the migration and antitumor efficacy of CAR T cells in treating solid tumors.

Morphological entropy encodes cellular migration strategies on multiple length scales

Cell migration is crucial for numerous physiological and pathological processes. A cell adapts its morphology, including the overall and nuclear morphology, in response to various cues in complex microenvironments, such as topotaxis and chemotaxis during migration. Thus, the dynamics of cellular morphology can encode migration strategies, from which diverse migration mechanisms can be inferred. However, deciphering the mechanisms behind cell migration encoded in morphology dynamics remains a challenging problem. Here, we present a powerful universal metric, the Cell Morphological Entropy (CME), developed by combining parametric morphological analysis with Shannon entropy. The utility of CME, which accurately quantifies the complex cellular morphology at multiple length scales through the deviation from a perfectly circular shape, is illustrated using a variety of normal and tumor cell lines in different in vitro microenvironments. Our results show how geometric constraints affect the MDA-MB-231 cell nucleus, the emerging interactions of MCF-10A cells migrating on collagen gel, and the critical transition from proliferation to invasion in tumor spheroids. The analysis demonstrates that the CME-based approach provides an effective and physically interpretable tool to measure morphology in real-time across multiple length scales. It provides deeper insight into cell migration and contributes to the understanding of different behavioral modes and collective cell motility in more complex microenvironments.

Site-specific regulation of Th2 differentiation within lymph node microenvironments.

T helper 2 (Th2) responses protect against pathogens while also driving allergic inflammation, yet how large-scale Th2 responses are generated in tissue context remains unclear. Here, we used quantitative imaging to investigate early Th2 differentiation within lymph nodes (LNs) following cutaneous allergen administration. Contrary to current models, we observed extensive activation and "macro-clustering" of early Th2 cells with migratory type-2 dendritic cells (cDC2s), generating specialized Th2-promoting microenvironments. Macro-clustering was integrin-mediated and promoted localized cytokine exchange among T cells to reinforce differentiation, which contrasted the behavior during Th1 responses. Unexpectedly, formation of Th2 macro-clusters was dependent on the site of skin sensitization. Differences between sites were driven by divergent activation states of migratory cDC2 from different dermal tissues, with enhanced costimulatory molecule expression by cDC2 in Th2-generating LNs promoting prolonged T cell activation, macro-clustering, and cytokine sensing. Thus, the generation of dedicated Th2 priming microenvironments through enhanced costimulatory molecule signaling initiates Th2 responses in vivo and occurs in a skin site-specific manner.

Steroid hormone signaling synchronizes cell migration machinery, adhesion and polarity to direct collective movement.

Migratory cells - either individually or in cohesive groups - are critical for spatiotemporally regulated processes such as embryonic development and wound healing. Their dysregulation is the underlying cause of formidable health problems such as congenital abnormalities and metastatic cancers. Border cell behavior during Drosophila oogenesis provides an effective model to study temporally regulated, collective cell migration in vivo. Developmental timing in flies is primarily controlled by the steroid hormone ecdysone, which acts through a well-conserved, nuclear hormone receptor complex. Ecdysone signaling determines the timing of border cell migration, but the molecular mechanisms governing this remain obscure. We found that border cell clusters expressing a dominant-negative form of ecdysone receptor extended ineffective protrusions. Additionally, these clusters had aberrant spatial distributions of E-cadherin (E-cad), apical domain markers and activated myosin that did not overlap. Remediating their expression or activity individually in clusters mutant for ecdysone signaling did not restore proper migration. We propose that ecdysone signaling synchronizes the functional distribution of E-cadherin, atypical protein kinase C (aPKC), Discs large (Dlg1) and activated myosin post-transcriptionally to coordinate adhesion, polarity and contractility and temporally control collective cell migration.