Abstract Purpose: This study aims to elucidate the molecular mechanisms driving malignant transformation from pre-malignant lesions, leveraging a CRISPR-engineered human retinoblastoma retinal organoid (RBRO) model that recapitulates the cone cell-of-origin and timing of multi-step retinoblastoma genesis. Background: Most retinoblastomas arise from maturing cone photoreceptor precursors (CPs) following biallelic RB1 inactivation. The process can be recapitulated in explanted fetal retina, where pRB-depleted CPs proliferate, followed by a 3-5 month indolence phase and emergence of retinoblastoma-like masses at tissue ages mirroring in vivo disease. CRISPR engineered retinal organoids (ROs) provide a promising model with which to define mechanisms that underlie malignant progression. Methods: We generated RB1 knockout iPSC lines through CRISPR editing of the GNAT2-EGFP cone reporter (Bai et al., PMID 37902188). Chimeric RBROs were produced by mixing RB1 knockout and unedited parental iPSCs. Proliferation dynamics, cell identities and cell state changes of EGFP+ RB1−/- cones were evaluated by live imaging of hydrogel-embedded RBROs, immunofluorescent (IF) staining, and deep full-length scRNA-seq. Additional CRISPR edits were introduced to test candidate drivers of indolence entry and escape. Results: In RB1 WT ROs, EGFP specifically, robustly and innocuously labeled post-mitotic cones. In RBROs, live imaging and IF staining revealed initial EGFP+ RB1−/- cone proliferation followed by a pre-malignant indolence phase starting at ∼d120. Most of the initially proliferating cones become Ki67-negative, with some adopting mature cone morphology. Retinoblastoma-like foci composed of cells co-expressing EGFP, cone markers, and Ki67 formed after ∼d280, a tissue age that equates to the first post-natal month when retinoblastomas emerge. Single-cell transcriptomic profiling at different tumorigenesis stages revealed distinct RB1−/- cell proliferation, cell differentiation, and cell stress states with high expression of CDKN2AARF RNA and p14ARF protein. In contrast to initially proliferating pre-indolence CPs, the later proliferating post-indolence retinoblastoma-like cells expressed CDKN2AARF RNA but not p14ARF protein, consistent with post-transcriptional silencing of p14ARF as a mechanism underlying indolence escape. RBROs lacking p14ARF exhibited sustained initial cone proliferation similar to indolence-escaped p14ARF-WT RBROs. Conclusion: We established a human retinoblastoma organoid model that recapitulates the cell-of-origin, developmental context, and temporal sequence of multi-step retinoblastoma genesis. This system uncovers stage-specific molecular signatures and highlights p53 pathway regulation – particularly p14ARF post-transcriptional silencing – as a key driver of the indolence-malignancy transition. Citation Format: Jinlun Bai, David S. Koos, Kevin Stachelek, Bhavana Bhat, Susan Asatrian, Patrick Belen, Sunjum Sanghari, Kayla Stepanian, Scott Fraser, Rex A. Moats, David Cobrinik, . Post-transcriptional silencing of p14ARF drives retinoblastoma malignant conversion in CRISPR-engineered retinal organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6182.

Abstract Introduction: Wilms tumor (WT) represents approximately 90% of pediatric renal malignancies. While standard treatments are effective for many patients, those with relapse or unfavorable histopathology experience poor survival and severe long-term toxicities. The extracellular matrix (ECM) plays a critical role in driving cancer progression and drug resistance. Our comprehensive profiling of WT revealed type II collagen alpha 1 (COL2A1), absent in normal kidney, to be highly expressed in high-risk tumors, highlighting its potential as a novel biomarker and therapeutic target. Methods: We utilized WT cancer stem cell (CSC)-like progenitors, 3D cultures, and patient-derived xenografts (PDXs) with metastatic and chemoresistant phenotypes to investigate COL2A1-mediated signaling. Comparative transcriptomics were performed on WT CSCs cultured on WT-versus normal kidney-derived decellularized ECM. Functional assays assessed epithelial-mesenchymal transition (EMT), tumor suppressor expression, and chemotherapy response following COL2A1 inhibition. Results: COL2A1-enriched substrates activated AKT signaling, induced EMT, downregulated tumor suppressors, and promoted chemoresistance. Transcriptomic analysis revealed COL2A1 upregulation alongside its transcriptional regulator SP1, correlating with enhanced tumorigenic pathways and reduced drug sensitivity. Notably, COL2A1 blockade using a specific antibody reversed EMT (i.e., increased cytokeratin and decreased vimentin), restored tumor suppressor expression, and enhanced chemotherapy response. Conclusions: COL2A1 is a critical ECM component driving therapy resistance in WT. Targeting COL2A1 offers a promising ECM-directed strategy to overcome treatment resistance and improve outcomes in high-risk WT patients. These findings highlight ECM-directed interventions as a new frontier in the treatment of pediatric kidney cancer. Citation Format: Wilson Yeung, Hripsime Chomoyan, Matthew E. Thornton, David S. Koos, Valentina Villani, Justin Sunwoo, Brendan H. Grubbs, Roger E. De Filippo, Laura Perin, Astgik Petrosyan. Targeting the extracellular matrix in Wilms tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7478.

Metallic nanoparticles (NPs) enhance radiotherapy through photoelectric absorption and Auger electron cascades, yet the effective spatial range over which these low-energy electrons induce biological damage remains poorly defined. Quantifying nanoscale energy deposition is essential for rational therapeutic design and safe clinical translation. Here, we establish a self-assembled polyelectrolyte-nanoparticle-cell architecture enabling nanometer-precision control of NP-cell separation (25-100 nm) to directly probe distance-dependent radiation enhancement. Layer-by-layer assembly produced uniform interfaces confirmed by spectroscopy, ellipsometry, electron microscopy, atomic force microscopy, and microgravimetry. Using human microglial (HMC3) and diffuse intrinsic pontine glioma (SU-DIPG-IV) cells, we quantified intracellular reactive oxygen species generation and γH2AX-marked DNA double-strand breaks following 137Cs γ-irradiation. Cells positioned 25.9 nm from the NP layer exhibited significantly increased DNA damage relative to NP-free controls, whereas damage progressively decreased with increasing separation, yielding a 250% differential effect between 25.9 and 97.5 nm. Modality-dependent attenuation profiles were observed across γ-ray, X-ray, and electron irradiation. These findings define the effective nanoscale interaction radius governing NP-mediated Auger enhancement and establish a technique for the interrogation of light-matter interactions for therapeutic energy deposition.

Kelp, brown macroalgae in the order Laminariales, provide ecosystem services vital to ocean biodiversity. However, kelp forests worldwide are declining due to abiotic stressors such as ocean warming. In this study, we present results from high-resolution confocal microscopy and in vivo imaging system imaging using protocols developed to visualize kelp gametophyte cells exposed to heat-stress treatments. Imaging revealed chloroplast mislocalization, fragmentation, and subsequent loss of chloroplasts in heat-stressed gametophyte cells. Additionally, nuclei exhibited fragmentation and a progressive loss of fluorescent signal, and the associated microbiome proliferated under various heat-stress treatments. Notably, because brown algae possess a continuous outer membrane that connects the nuclear envelope and the chloroplast envelope, these observations suggest a cellular vulnerability underlying thermal sensitivity in brown macroalgae. Finally, by comparing heat-stress tolerant and heat-stress sensitive genotypes, we found that genotypes with higher heat tolerance exhibited substantially fewer abnormalities compared to sensitive ones.

It has become evident from decades of clinical trials that multimodal therapeutic approaches with focus on cell intrinsic and microenvironmental cues are needed to improve understanding and treat the rare, inoperable, and ultimately fatal diffuse intrinsic pontine glioma (DIPG), now categorized as a diffuse midline glioma. In this study we report the development and characterization of an in vitro system utilizing 3D Tumor Tissue Analogs (TTA), designed to replicate the intricate DIPG microenvironment. The innate ability of fluorescently labeled human brain endothelial cells, microglia, and patient-derived DIPG cell lines to self-assemble has been exploited to generate multicellular 3D TTAs that mimic tissue-like microstructures, enabling an in- depth exploration of the spatio-temporal dynamics between neoplastic and stromal cells. The 3D-TTA model recapitulates clinical patterns of DIPG growth, evidenced by resistance to chemotherapy, HDAC and proteasome inhibitors, as well as sensitization to the antibody-activated innate immune microenvironment including complement proteins and surrounding microglia. Multimodal fluorescence imaging platforms integrated with high-throughput omics revealed that alterations in tumor cell motility and growth in the 3D-TTA model compared to tumor cell only spheroids correlated with specific transcriptomic and proteomic changes. STAT3, ITGA5, LGALS1, SOD2, MVP, and CLIC1, associated with microenvironment signaling, DNA replication, and immune regulation, were identified as potential novel targets in the 3D model. The results indicate that the 3D TTA platform developed here represents a powerful tool for preclinical studies, paving the way for identification/validation of tissue specific biomarkers and novel drug targets, thus advancing disease management strategies for DIPG in children.

This study aims to elucidate the molecular mechanisms driving the transition from pre-malignant lesions to malignancy using a human retinoblastoma retinal organoid (RBRO) model that faithfully recapitulates the cell-of-origin and multi-step retinoblastoma genesis. Retinoblastoma originates from maturing cone photoreceptor precursors with biallelic RB1 inactivation. In explanted fetal retinae, pRB-depleted post-mitotic cone precursors proliferate, followed by a 3-5 month premalignant indolence phase before retinoblastoma-like masses emerge at tissue ages mirroring in vivo disease. Research on this transition is limited by fetal tissue availability, but CRISPR engineered retinal organoids (ROs) provide a promising alternative. This study introduces a robust organoid platform to model and investigate the retinoblastoma indolence-malignancy transition. We generated cone-reporter iPSC lines through CRISPR knock-in of EGFP-P2A at the GNAT2 locus. A second round of CRISPR editing produced homozygous RB1 knockout. Chimeric RB1+/+ ROs and RB1-/- RBROs were generated from edited iPSCs mixed with unedited parental iPSCs. ROs and RBROs were embedded in hydrogel and live-imaged episodically to track EGFP+ cone proliferation dynamics. Deep full-length scRNA-seq was carried out on FACS isolated EGFP+ RB1+/+ and RB1-/- cones at key developmental and tumorigenesis stages, with results validated by immunofluorescent staining. In RB1 WT ROs, EGFP specifically, robustly and innocuously labeled immature and mature cones. In RBROs, bi-weekly live confocal imaging revealed initial EGFP+ RB1-/- cone proliferation followed by a pre-malignant indolence phase starting at ∼d150. The majority of the initially proliferating cones were Ki67-negative with some adopting mature cone morphology. Nascent retinoblastoma-like foci co-expressing EGFP, cone markers, and Ki67 formed after ∼d280, a tissue age that equates to the first post-natal month when early retinoblastomas typically emerge. Single cell transcriptomics of RB1-/- cones from multiple tumorigenesis stages showed distinct molecular signatures of proliferation, differentiation and stress cell states and suggest a role of p53 pathway in indolence entry and escape. Immunofluorescent staining suggested post-transcriptional silencing of p14ARF underlying indolence escape. We established a human retinoblastoma organoid model that recapitulates the cell-of-origin and timing of multi-step retinoblastomagenesis. This model revealed distinct molecular signatures at each tumorigenesis stage and highlighted altered expression of a p53 pathway regulator during the retinoblastoma indolence-malignancy transition. Jinlun Bai, David S. Koos, Kayla Stepanian, Kevin Stachelek, Bhavana Bhat, Scott E. Fraser, Rex A. Moats, David Cobrinik. Deciphering retinoblastoma indolence-malignancy transition with RB1 knockout cone reporter retinal organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3944.

Abstract Purpose: This study aims to develop a human retinoblastoma organoid (RBRO) model that recapitulates the cell-of-origin and multi-step retinoblastoma genesis. Background: Retinoblastomas originate from maturing cone photoreceptor precursors with biallelic RB1 inactivation. Rb1 mutant animal models fail to recapitulate retinoblastomagenesis with a cone precursor cell-of-origin, likely due to human-specific cone development features. In explanted fetal retina, pRB-depleted post-mitotic cone precursors proliferate, followed by a 3-5 month premalignant indolence phase before retinoblastoma-like masses emerge at tissue ages similar to retinoblastomas in vivo. However, tissue availability limits research with this disease model. RB1−/- retinal organoids (ROs) provide a potential alternative, as they demonstrate cone proliferation, but they deteriorate before forming indolent premalignant lesions or malignant retinoblastoma foci. Here, we developed methods with which to examine the effects of cone precursor pRB loss in otherwise healthy retinal tissue. Methods: We generated cone-reporter GNAT2-EGFP iPSC lines through CRISPR knock-in of EGFP-P2A at the N-terminus of GNAT2 in WTC11-mTagRFPT-LMNB1. A second round of CRISPR editing produced homozygous RB1 knockout. Chimeric RB1 WT ROs and RB1-null RBROs were generated from cone reporter iPSCs mixed with unedited parental iPSCs. ROs and RBROs were embedded in hydrogel and live-imaged episodically to track EGFP+ cone proliferation dynamics. scRNA-seq was carried out on FACS isolated EGFP+ RB1-null cones at various ages. Results: In RB1 WT ROs, EGFP+ cone precursors appeared at d34 and adopted cone morphology at ~d120, with rapid inner segment growth between ~d120 and ~d150. Immunohistochemistry confirmed cone-specific EGFP expression. In RB1-null RBROs, bi-weekly live confocal imaging revealed initial EGFP+ RB1−/− cone proliferation followed by a pre-malignant indolence phase starting at ~d150. The majority of the initially proliferating cones were Ki67-negative with some adopting mature cone photoreceptor morphology. Nascent retinoblastoma-like foci co-expressing EGFP, cone markers, and Ki67 formed in five of six RBROs after d281, a tissue age that equates to the first post-natal month when early retinoblastomas typically emerge. scRNA-seq of EGFP+ RB1−/− cones from the initial proliferation, indolence, and retinoblastoma-like stages revealed distinct molecular signatures. Conclusions: We generated a human retinoblastoma organoid model that recapitulates the cell-of-origin and timing of multi-step retinoblastomagenesis, with each tumorigenesis stage harboring distinct molecular signatures. This model may enable the identification of epigenetic and transcriptomic changes underlying malignant progression and screening for compounds that efficiently block this process. Citation Format: Jinlun Bai, David S. Koos, Kayla Stepanian, Kevin Stachelek, Bhavana Bhat, Scott Fraser, Rex Moats, David Cobrinik. Modeling multi-step retinoblastoma genesis with cone reporter retinal organoids [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 236.

Kelps are vital for marine ecosystems, yet the genetic diversity underlying their capacity to adapt to climate change remains unknown. In this study, we focused on the kelp Macrocystis pyrifera a species critical to coastal habitats. We developed a protocol to evaluate heat stress response in 204 Macrocystis pyrifera genotypes subjected to heat stress treatments ranging from 21 degrees C to 27 degrees C. Here we show that haploid gametophytes exhibiting a heat-stress tolerant (HST) phenotype also produced greater biomass as genetically similar diploid sporophytes in a warm-water ocean farm. HST was measured as chlorophyll autofluorescence per genotype, presented here as fluorescent intensity values. This correlation suggests a predictive relationship between the growth performance of the early microscopic gametophyte stage HST and the later macroscopic sporophyte stage, indicating the potential for selecting resilient kelp strains under warmer ocean temperatures. However, HST kelps showed reduced genetic variation, underscoring the importance of integrating heat tolerance genes into a broader genetic pool to maintain the adaptability of kelp populations in the face of climate change.

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology I (PD16)1 May 2024PD16-06 EXTRACELLULAR MATRIX IN THE TUMOR MICROENVIRONMENT AND ITS INFLUENCE ON CANCER David S. Koos, Xiaogang Hou, Esteban Fernandez, Matthew E. Thornton, Brendan H. Grubbs, Roger E. De Filippo, Stefano Da Sacco, Laura Perin, and Astgik Petrosyan David S. KoosDavid S. Koos , Xiaogang HouXiaogang Hou , Esteban FernandezEsteban Fernandez , Matthew E. ThorntonMatthew E. Thornton , Brendan H. GrubbsBrendan H. Grubbs , Roger E. De FilippoRoger E. De Filippo , Stefano Da SaccoStefano Da Sacco , Laura PerinLaura Perin , and Astgik PetrosyanAstgik Petrosyan View All Author Informationhttps://doi.org/10.1097/01.JU.0001009560.23593.56.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The tumor microenvironment's extracellular matrix (ECM) is critical in supporting normal and cancerous cell growth and development. In Wilms Tumor (WT), a pediatric renal cancer that arises from abnormal kidney development, we investigate the influence of the ECM on cellular behavior. We use a combination of decellularization techniques with two-photon excited fluorescence (TPEF) microscopy and transcriptomics to identify how cancer ECM vs. normal kidney ECM influences normal and cancer cells. METHODS: We decellularized normal kidneys and WT patient samples using an optimized decellularization technique. Second-harmonic generation (SHG) microscopy and two-photon excited fluorescence (TPEF), combined with immunohistochemistry, were used to characterize the decellularized matrices (dECM). Expression of various cancer-related proteins in WT dECM samples was compared to normal kidney dECM samples using the Proteome Profiler Human XL Oncology Array. Changes in gene expression of seeded WT and human fetal kidney (hFK) nephron progenitor cells on different dECM scaffolds were examined by immunofluorescence and bulk RNA-seq. RESULTS: Tumor ECM imaged at 700 um in-depth shows a difference in fiber area and angle, with the outer 250 um layer comprising dense and elongated fibers, followed by pocketed and mesh-like structures when imaged at 300 um in-depth. Analysis of tumor ECM vs. normal kidney ECM showed high expression of several oncoproteins, such as ERB2/3, PAI-1, and reduced CCL2 and BCL-X expression. Cellular behavior and transcriptomic profiles were altered when cultured on normal vs. tumor ECM for 21 days. RNA-Seq data of tumor cells seeded on tumor ECM vs. normal kidney ECM expressed Gene Ontology (GO) pathways favoring proliferation and reduction of pathways favoring differentiation. Normal cells seeded on tumor ECM vs. normal kidney ECM showed predicted activation of cancer development pathways and inhibition of apoptotic pathways. CONCLUSIONS: This study provides valuable insights into the role of the ECM in regulating cancer cell behavior. These findings have significant future implications for developing physiologically relevant in vitro tumor models and identifying novel therapeutic targets and mechanisms against tumor development and progression targeting the ECM. Source of Funding: Children's Hospital Los Angeles Intramural RCDA 2023 The GOFARR Research Fund © 2024 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 211Issue 5SMay 2024Page: e367 Advertisement Copyright & Permissions© 2024 by American Urological Association Education and Research, Inc.Metrics Author Information David S. Koos More articles by this author Xiaogang Hou More articles by this author Esteban Fernandez More articles by this author Matthew E. Thornton More articles by this author Brendan H. Grubbs More articles by this author Roger E. De Filippo More articles by this author Stefano Da Sacco More articles by this author Laura Perin More articles by this author Astgik Petrosyan More articles by this author Expand All Advertisement PDF downloadLoading ...

Abstract Kelps are vital for marine ecosystems, yet the genetic diversity underlying their capacity to adapt to climate change remains unknown. In this study, we focused on the kelp Macrocystis pyrifera (M. pyrifera), a species critical to coastal habitats. We developed a phenotyping protocol to evaluate heat stress response in 204 M. pyrifera gametophytes, subjected to heat stress treatments ranging from 21°C to 27°C. Here we show that genotypes exhibiting a heat-stress tolerant (HST) phenotype as haploid gametophytes also produced greater biomass as diploid sporophytes in a warm-water ocean farm. This correlation suggests a predictive relationship between early-stage HST and later-stage growth performance, indicating the potential for selecting resilient kelp strains under warmer ocean temperatures. However, HST kelps showed reduced genetic variation, underscoring the importance of integrating heat tolerance genes into a broader genetic pool to maintain the adaptability of kelp populations in the face of climate change.

Fluorescent reporter pluripotent stem cell (PSC) derived retinal organoids are powerful tools to investigate cell type-specific development and disease phenotypes. When combined with live imaging, they enable direct and repeated observation of cell behaviors within a developing retinal tissue. Here, we generated a human cone photoreceptor reporter line by CRISPR/Cas9 genome editing of WTC11-mTagRFPT-LMNB1 human induced pluripotent stem cells (iPSCs) by inserting enhanced green fluorescent protein (EGFP) coding sequences and a 2A self-cleaving peptide at the N-terminus of Guanine Nucleotide-Binding Protein Subunit Alpha Transducin 2 (GNAT2). In retinal organoids generated from these iPSCs, the GNAT2-EGFP allele robustly and exclusively labeled both immature and mature cones starting at culture day 34. Episodic confocal live imaging of hydrogel immobilized retinal organoids allowed tracking of morphological maturation of individual cones for >18 weeks and revealed inner segment accumulation of mitochondria and growth at 12.2 cubic microns per day from day 126 to day 153. Immobilized GNAT2-EGFP cone reporter organoids provide a valuable tool for investigating human cone development and disease.

During adolescence, the lungs expand while alveoli increase greatly in number. A precise and deep understanding of the biological processes responsible for the many fold increase in alveoli during adolescence could form the foundation for novel treatments to reoptimize lung function after disease or injury. Herein we provide a unique insight into the alveolar growth in the mouse lung during first postnatal 4 weeks herein described as the adolescence phase. Using 3-Dimensionnal (3D) high-resolution large field of view optical imaging coupled with qualitative and quantitative analysis, we suggest the bulk of alveolarization during adolescent lung growth occurs while the surface to volume of the acinar volume remains constant. The most distal 10 or more bifurcations within the acinar saccular region, comprised of small ducts and alveoli, are of similar scale. We explored the connections between the growth of lung and it’s structural and functional parameters, by comparing the correlations between alveolar size, surface area of gas exchange regions, breathing rate, metabolic rate, and oxygen demand across mammalian species which exhibit widely divergent body sizes.

Herein we show, using several novel imaging and computational approaches, how the air exchange units (AEUs) of the lung develop from the tips and sides of distinct families of tortuous ducts, that themselves ramify as distinct families distal to the bronchoalveolar duct junctions (BADJs), prenatally in humans but postnatally in mice. The mature AEUs thus consist of indented spheroids tightly packed between quite regularly spaced distal ducts. Since the diameter of the BADJs and the distal ducts increases rather than decreasing during the formation of AEUs, we further deduce that the AEUs must form by circular epithelial precursor buckling at their mouths with resulting extrusion of their lumen into the surrounding mesenchyme, stabilized firstly by interlocking rings of elastin and later by rings of elastin and collagen fibers surrounding the mouth of each of the prospective AEUs. Furthermore, we show that the surface of each of the AEUs is highly rugose, being indented by the capillary network that lies close beneath the AES membranes. We propose that, the kissing theorem proposed of Newton that expresses the number of times billiard balls may touch within their frame is a parsimonious solution to achieving optimum packing of the distal AES unit spaces, while allowing sufficient space between them to allow for conducting airways, closely applied pulsatile capillary blood vessels, lymphatics, nerves and other key components of the interstitial mesenchyme. Summary statement Employing novel imaging and computational approaches we here deduce some new concepts as to how the distal shaping of airway lineage stem and progenitor cells may contribute to the growth and form of the air exchange surface (AES) of the lung, distal to the bronchoalveolar duct junction (BADJ). We then propose that the AES extrudes from the sides and tips of distinct families of small ducts, apparently by buckling of the thinning luminal epithelium into the surrounding mesenchyme, stabilized firstly by rings of elastin and later by rings of collagen and elastin fibers, that surround the mouth of each distal unit of the AES. We propose this mechanism as a parsimonious solution to achieving the optimum form, packing density and functional efficiency of the AES, while allowing sufficient space between for the plumbing of conducting airways, pulsatile capillary blood vessels, lymphatics, nerves and other key matrix and cellular components within the interstitial mesenchyme.