Multiple cell types have been implicated in pathogenesis of pulmonary fibrosis with pericytes emerging as a new focus due to their role in promoting fibrotic remodeling. Fibrotic environment changes normal pericyte functions, but transcriptional programs regulating pericyte transition towards fibrotic state remain unclear. Utilizing single cell RNA-sequencing of human pulmonary fibrotic lungs and mouse genetic models, we identified a unique cluster of fibrosis-associated pericytes which was not present in normal lungs and exhibited a distinctive transcriptional signature indicating transition of normal pericytes to a fibrotic state. FOXF1 was identified as one of the transcription factors decreased in fibrotic pericytes. Pericyte-specific deletion of Foxf1 increased severity of pulmonary fibrosis in bleomycin mouse model as demonstrated by reduced survival, impaired lung functions, increased body weight loss and increased fibrotic remodeling. Pericyte-specific overexpression of Foxf1 attenuated pulmonary fibrosis, reversed fibrotic changes and improved survival outcomes. Based on single cell RNA-sequencing and chromatin immunoprecipitation sequencing, FOXF1 transcriptionally regulates an extensive pericyte signaling network critical for pulmonary fibrosis. In vitro, FOXF1 transcriptionally activated ID3 which inhibited fibroblast activation through decreased secretion of IL8 and CXCL1 by pericytes. Altogether, FOXF1 prevents transition of pericytes to a fibrotic state, suggesting new therapeutic opportunities for treatment of pulmonary fibrosis.

Abstract How intestinal stem cells (ISCs) are regulated during inflammation remains largely unexplored, leading to a lack of effective treatments for inflammatory bowel diseases (IBD). ISC-mediated intestinal epithelial regenerative repair can be regulated by intra-epithelial T lymphocytes, whose maturation is controlled by STAT5 dimeric or tetrameric activation. However, the mechanisms by which the T lymphocytes can protect ISCs are unclear. Here we hypothesize that tetrameric STAT5 regulates intra-crypt T cells to act as niche cells for ISC regeneration. Using IBD biospecimens, STAT5-hyperactive, tetramer-deficient mice and organoids, we found IBD–ulcerative colitis exhibited more crypt TCRγδ+STAT5+ T cells and less ISC pluripotency than healthy patients. Compared with wild-type mice, depleting tetrameric STAT5 in mice significantly increased ISC-mediated intestinal epithelial hyperplasia, TCR gene signatures, crypt TCRγδ+ T cells with elevated STAT5 tyrosine phosphorylation (pYSTAT5) and IL-17A levels, amplified both Lgr5hi and Lgr5low ISC proliferation and promoted de novo crypt regeneration with increased TCRγδ+ cell influx post irradiation or colitis. By contrast, depleting tetrameric STAT5 in organoids reduced ISC pluripotency and organoid growth post irradiation. Mechanistically, chromatin immunoprecipitation and single-cell RNA sequencing analyses with crypt cells revealed that depleting STAT5 tetramers decreased STAT5-binding on the Metallothionein 1 (Mt1) locus in crypt T cells and increased Mt1 expression, which leads to T cell migration into crypts and enhanced ISC regeneration. Together, the tetrameric STAT5 suppresses the formation of the crypt T cell niche. Interrupting STAT5 tetramers promotes the expansion of crypt TCRγδ cells, providing a target for promoting ISC regenerative repair during IBD–ulcerative colitis.

The pulmonary system is a vital interface between the body and the external environment, making it highly vulnerable to environmental, infectious, and genetic insults. Precision nanomedicine offers a promising strategy to overcome the limitations of conventional gene and drug therapies, including safety concerns associated with viral vectors, instability of therapeutic agents, suboptimal cellular internalization, and a critical lack of tissue- and cell-specific targeting. Nanoparticle-based delivery platforms address these challenges by enhancing therapeutic stability and bioavailability, enabling controlled release, facilitating cellular uptake and endosomal escape, and achieving targeted delivery to specific lung compartments. While recent literature often focuses on specific nanoparticle types or isolated pathologies, this work provides a comprehensive overview of the current state of respiratory nanomedicine, bridging fundamental nanoparticle bioengineering with a wide range of pulmonary pathologies and the obstacles to clinical translation. We discuss the key physicochemical properties of nanoparticles for pulmonary biomedical applications, along with advanced design strategies for targeted delivery. Given the unique architecture and physiology of the lung, we compare the advantages and limitations of pulmonary versus systemic administration routes, emphasizing context-specific delivery strategies. Nanoparticle design and therapeutic applications are explored across a broad spectrum of diseases, including pulmonary fibrosis, chronic obstructive pulmonary disease, infections, pulmonary vascular disease, cystic fibrosis, asthma, lung cancers, and neonatal pulmonary disorders. Finally, we evaluate the current status of clinical trials, highlighting translational challenges such as biological barriers, long-term safety, and manufacturing. Future perspectives and interdisciplinary strategies are proposed to advance the clinical translation of nanocarriers for respiratory diseases.

Despite the initial responses to the tyrosine kinase inhibitor (TKI) for cancer therapy, many patients often relapse with no curative regimens available. Further, the ability to target therapeutic agents to cancer cells with appropriate doses remains challenging in the clinic, especially for leukemia. Here, we show that naïve CML cells are dynamically heterogeneous in colony formation. Larger clones expand while smaller ones diminish and eventually disappear. Compared to resistant cells, parental populations, including CD44+ stem cells, form a greater number of larger, solid spheroids. Upregulation of fat mass and obesity associated protein (FTO), an RNA N6-methyladenosine demethylase, and stem cell markers (e.g., CD44, CD133, CD25) is more obvious in resistant cells compared to parental cells. FTO inhibitors (e.g., CS1, FB23-2) appreciably impair the growth of resistant cells either alone or in combination with nilotinib. FTO protein expression is unexpectedly upregulated by CS1 or FB23-2 treatment in multiple leukemia cell lines. We then constructed RNA nanoparticles encapsulating FTO siRNAs and conjugated with anti-CD133 RNA aptamers. We showed that, compared to negative control, these nanoparticles were taken up much more efficiently by resistant cells that highly express CD133. Treatment with the CD133-guided FTO siRNA nanoparticles efficiently silenced FTO expression in resistant cells, which leads to a significant reduction in their colony and spheroid formation. These findings offer new insights into cancer drug resistance and advance the application of RNA nanotechnology for treating leukemia. The research provides a foundation for developing novel, targeted therapies for resistant leukemia.

Lung cancer remains the leading cause of cancer-related mortality worldwide, with conventional treatments often limited by systemic toxicity, different tumor sensitivity to the drugs, and the emergence of multidrug resistance. To address these challenges, nanoparticle-based delivery systems have emerged as an innovative strategy, enabling the simultaneous transport of multiple agents, including chemotherapeutic drugs and expression vectors, to enhance treatment efficacy and overcome tumor resistance. This review explores various nanocarrier platforms, such as liposomes, solid lipid nanoparticles, polymeric micelles, and inorganic nanoparticles, specifically designed for lung cancer therapy. Synergistic effects and physicochemical properties of therapeutic agents must be carefully considered in the design of nanoparticle-based co-delivery systems for lung cancer therapy. We highlight the applications of these nanoparticle systems in drug–drug, gene–gene, and drug–gene co-delivery approaches. By addressing the limitations of traditional therapies, nanoparticle-based systems offer a promising avenue to improve outcomes in patients with lung cancers.

Cancer cells re-program normal lung endothelial cells (EC) into tumor-associated endothelial cells (TEC) that form leaky vessels supporting carcinogenesis. Transcriptional regulators that control the reprogramming of EC into TEC are poorly understood. We identified Forkhead box F1 (FOXF1) as a critical regulator of EC-to-TEC transition. FOXF1 was highly expressed in normal lung vasculature but was decreased in TEC within non-small cell lung cancers (NSCLC). Low FOXF1 correlated with poor overall survival of NSCLC patients. In mice, endothelial-specific deletion of FOXF1 decreased pericyte coverage, increased vessel permeability and hypoxia, and promoted lung tumor growth and metastasis. Endothelial-specific overexpression of FOXF1 normalized tumor vessels and inhibited the progression of lung cancer. FOXF1 deficiency decreased Wnt/β-catenin signaling in TECs through direct transcriptional activation of Fzd4. Restoring FZD4 expression in FOXF1-deficient TECs through endothelial-specific nanoparticle delivery of Fzd4 cDNA rescued Wnt/β-catenin signaling in TECs, normalized tumor vessels and inhibited the progression of lung cancer. Altogether, FOXF1 increases tumor vessel stability, and inhibits lung cancer progression by stimulating FZD4/Wnt/β-catenin signaling in TECs. Nanoparticle delivery of FZD4 cDNA has promise for future therapies in NSCLC.

Endothelial cell dysfunction occurs in a variety of acute and chronic pulmonary diseases including pulmonary hypertension, viral and bacterial pneumonia, bronchopulmonary dysplasia, and congenital lung diseases such as alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). To correct endothelial dysfunction, there is a critical need for the development of nanoparticle systems that can deliver drugs and nucleic acids to endothelial cells with high efficiency and precision. While several nanoparticle delivery systems targeting endothelial cells have been recently developed, none of them are specific to lung endothelial cells without targeting other organs in the body. In the present study, we successfully solved this problem by developing non-toxic poly(β-amino) ester (PBAE) nanoparticles with specific structure design and fluorinated modification for high efficiency and specific delivery of nucleic acids to the pulmonary endothelial cells. After intravenous administration, the PBAE nanoparticles were capable of delivering non-integrating DNA plasmids to lung microvascular endothelial cells but not to other lung cell types. IVIS whole body imaging and flow cytometry demonstrated that DNA plasmid were functional in the lung endothelial cells but not in endothelial cells of other organs. Fluorination of PBAE was required for lung endothelial cell-specific targeting. Hematologic analysis and liver and kidney metabolic panels demonstrated the lack of toxicity in experimental mice. Thus, fluorinated PBAE nanoparticles can be an ideal vehicle for gene therapy targeting lung microvascular endothelium in pulmonary vascular disorders.

Abstract Background The increased JAK2 and EZH2 plays a critical role in the colorectal or colon cancer (CRC) progression. Targeting aberrant JAK2 expression caused by JAK2 gain-of-function (GOF) mutation, JAK2V617F and deregulated EZH2 kinase activity, JAK2 and/or EZH2 inhibitors have been used to treat the advanced CRCs. However, it is unclear whether the aberrant JAK2 can directly induce CRC or intestinal epithelial (IEC) malignancy. We hypothesize that the overexpressed JAK2 driven by JAK2V617F GOF mutation increases IEC neoplasia in which depletion of EZH2 can lead to IEC malignant neoplasm. Materials and Methods Colorectal samples were collected from 20 cases of CRC patients. Immunohistochemistry staining (IH), confocal immunofluorescence staining (IF) and bulk RNAseq were used to determine LGR5, Ki67, JAK2, EZH2, mutant β-catenin and JAK2V617F GOF mutation. Crypt-derived organoids or tumoroids were used to determine the JAK2 and EZH2 in the proliferative LGR5 cells (Ki67hiLGR5+) and were treated with JAK2 and/or EZH2 inhibitors. The mice with JAK2 and/or EZH2 deficiency in IECs were generated by crossing VilCreER mice, floxed-Jak2 and/or floxed-Ezh2 mice to determine the intestinal and colonic neoplasia. Lgr5, Ki67, Cdx2 Jak2, Ezh2, and mutant β-catenin IH or IF, organoids culture and peritoneal xenograft were used to determine the malignant neoplasm. The JAK2V617F-transfected HT29 or Caco-2 cells were used to determine the migration induced by JAK2V617F GOF mutation, then treated with EZH2 inhibitors to determine the effects of EZH2 inhibition on JAK2V617F GOF mutation-induced migration. The spheres were formed with Caco-cells with JAK2V617F GOF mutation and were used to observe the effects of JAK2V617F GOF mutation on IEC neoplasia formation. Results JAK2 and/or EZH2 overexpression are positively correlated with the probability of CRC initiation. JAK2 and EZH2 are highly expressed in the CRC tumoroids. Inhibition of EZH2 increases the CRC tumoroids growth and spheroid formation, and reduces budding numbers while reducing the proliferation of organoids from healthy crypts. Jak2 over-expression in IECs increases intestinal microadenoma, and depletion of EZH2 in the adenoma increases mutant β-catenin and severity of intestinal adenoma, suggesting that depletion of EZH2 enhances the malignancy of overexpressed Jak2-derived IEC neoplasia. Consistently, JAK2V617F GOF mutation increases HT29 or Caco-2 cell migration. Conclusion JAK2V617F GOF mutation can drive IEC neoplastic proliferation and adenoma, depletion of EZH2 in the adenoma leads to IEC malignant neoplasm. Our research suggests that loss of Ezh2 may synergistically cooperate with the Jak2V617F mutation in the pathogenesis of IEC malignant neoplasm. Citation Format: Nardana Esmaeili, Ahmed Bakheet, Wen Gao, Haifeng Li, Dan Cai, Xiaonan Han. Depletion of Ezh2 in the intestinal adenoma induced by JAK2V617F mutation leads to the malignant neoplasm [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 260.

STAT5B deficiency, a rare autosomal recessive disorder characterized by severe growth hormone insensitivity (GHI) and immunodeficiency, can manifest as fatal pulmonary complications. We describe atypical STAT5B deficiency associated with a novel homozygous frame-shift STAT5B variant [c.1453delG, p.(Asp485Thrfs*29)] identified in a young 17.6 yr old female subject who had severe postnatal growth impairment, biochemistries typical of GHI, an immune profile notable for hypergammaglobulinaemia and elevated B lymphocytes, and lack of pulmonary disease. Marked elevation of serum prolactin and pathologically diagnosed eczema were evident. In reconstitution studies, the STAT5B p.(Asp485Thrfs*29) was expressed although expression was reduced compared to wild-type STAT5B and a previously identified STAT5B p.(Gln368Profs*9) variant. Both truncated STAT5B peptides could not be activated by GH, nor mobilize to the nucleus. We conclude that an intact, func-tional, STAT5B is essential for normal GH-mediated growth, while expressed loss-of-function STAT5B variants may alleviate severe immune and pulmonary issues normally associated with STAT5B deficiency.

Background: Lgr5 intestinal stem cells (ISC) are augmented by colorectal (CRC), but it is not clear if the aberrant Lgr5 ISCs result in the metastatic phenotypes of CRC, suggesting that epistasis-involved mutations in CRC suppressor genes or oncogene control Lgr5 ISC transformation into cancer stem cells (CSC). Somatic mutations in STAT5A or B are highly associated with CRC. Cytokine-STAT5 action is essential for Lgr5 ISC stemness, and enhanced STAT5 phosphorylation can induce Lgr5 ISC differentiation into secretory niche cells. Using human and mouse tumoroids, and organoid xerograph models, we aim to determine the effects of STAT5 oncogenic activation on Lgr5 ISC malignant and metastatic phenotypes. Methods: Colorectal surgical specimen from CRC and hepatic metastatic CRC patients were collected to perform RNAseq or single cell RNAseq (scRNAseq) analysis. Compound-mutant mice with STAT5 loss or gain of function in Apcmin+/- mice, were generated by crossing Apcmin+/- mice with Lgr5CreER and Rs26CreER, and Stat5f/f (loss of function of Stat5) or icS5f/f (gain of function of STAT5A) mice. CRISPR/Cas9 gene editing was applied to engineer APC mutations in human epithelial cell lines or Apc mutations in Lgr5 ISCs. Apc-mutant Lgr5 ISCs or tumoroids from Apcmin+/- mice combined with STAT5 loss or gain of function were differentiated, then were under intra-abdominal xenotransplantation. APC-mutant intestinal epithelial cell lines were used to determined CSC migration in the presence of STAT5 activation. STAT5 inhibitors were used to treat Apcmin+/- mice or tumoroids. Results: Human subject studies revealed that hepatic metastatic CRC patients exhibited the robustly increased STAT5A and/or Lgr5 colonic cancer cells. Murine studies showed that constitutively STAT5A activation (Ca-pYSTAT5) in colonic Apcmin+/- CSCs promoted CSC migration, invasion and metastasis. In vivo migration assay showed that transient STAT5A activation in the APC-mutant intestinal epithelia accelerated the migration of APC-mutant epithelia. Ex Vivo tumoroid xenotransplantation showed that Ca-pYSTAT5 resulted in tumoroid invasion into liver. Lineage tracing analysis showed that Ca-pYSTAT5 expanded Lgr5 CSCs in tumoroids and drove Lgr5 CSCs to hepatic metastasis. Importantly, Stat5 deletion in Lgr5 ISCs abolishes colon cancer orgnoids invasion. Pharmacological inhibition of STAT5 dimerization leads to Lgr5 cancer organoid degeneration and partially annihilated intestinal adenoma in Apcmin+/-. CHIP-seq analysis showed that Ca-pYSTAT5 increased an aberrant high level of Bcl6 expression by inducing super-enhancer activation in the Bcl-6 locus. Conclusion: Aberrantly activated Cytokine-STAT5A signaling is required for ISC malignancy and CSC hepatic metastasis. STAT5 blockade could be important to impair CRC metastasis. Citation Format: Haifeng Li, Wen Gao, Ruixue Liu, Xiaonan Han. Transforming intestinal stem cells into metastatic cancer Stem cells by aberrantly activated cytokine-STAT5A signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2450.

Abstract Background: CDX2 is a transcription factor expressed in the gastro-intestinal (GI) epithelial (IEC) and stromal cells. CDX2 along with APC can regulate Lgr5 intestinal stem cell (ISC) differentiation to control GI development or neoplasia. CDX2 or APC loss function is associated with CRC advance. However, whether APC directly regulates CRC metastasis is not clear. We aim to determine the role of inactive Apc gene in Cdx2 GI cells on Lgr5 ISC metastasis. Methods: Tissue sections were collected from CRC or CRC hepatic-metastatic patients, CDX2, CTNNB1, α-SMA, APC and LGR5 expressions were examined. Apc flox mice were crossed with Lgr5CreER and/or Cdx2CreER mice to inactivate Apc gene in Lgr5 ISCs, Cdx2 IECs or stromal cells. Cdx2CreER;Apc mice were then crossed with tgfr2 flox mice to deplete tgfr2 in the Apc-deficient Cdx2 cells. GI tract and liver histology was evaluated and the expression of Lgr5, APC, α-SMA, β-catenin, and CDX2 were examined. The colorectal polyps induced by Apc inactivation were dissected, total RNA was extracted to perform RNA-Seq and quantitative PCR analyses. The colorectal organoids were differentiated and transplanted into abdominal cavity of recipient mice. Meanwhile, HT-29 cells were transfected with APC and/or CDX2 gRNAs. Results: CTNNB1 and LGR5 expression were increased in human CRC and hepatic-metastatic CRC while interstitial CDX2 and SMA colocalization were robustly pronounced compared to normal colorectum. TCGA analysis showed the positive correlation of APC lower and CDX2 higher with metastatic rectal cancer. Apc depletion in Lgr5 cells in 6-week-old mice led to Lgr5+ rectal adenocarcinoma and Lgr5 crypts in liver while depletion of Apc in Lgr5 cells in 3-month-old mice only resulted in Lgr5 intestinal adenoma. Apc depletion in Cdx2 cells led to rectal fibroma and intestinal adenoma as well as undifferentiated metastatic Lgr5 cells in liver. Notably, Apc depletion decreased crypt CDX2 while increased colocalization of CDX2 and α-SMA in the stromal cells of adenocarcinoma, and increased expression of TGFBβr2, β-catenin, and epithelial-mesenchymal transition (EMT) markers (Fibronectin and Vimentin). Finally, tgfr2 depletion in Apc-inactive Cdx2 cells impaired GI cancer cell metastasis to liver. Colorectal orgnoids exhibited cancer phenotypes upon Apc depletion in CDX2 or Lgr5 cells. Intra-abdominal xenotransplantation exhibited that organoids with Apc deficiency in Lgr5 cells appeared no colonization on or invasion of liver tissues whereas the organoids with Apc deficiency in Cdx2 cells invaded liver. Conclusion: Reduced Apc in Cdx2 cells are required for Lgr5 cell transformation to Lgr5- cancer stem cells. Inactive intestinal Apc increases Cdx2-α-SMA stromal niche cells and facilitates Lgr5 ISC metastasis to liver partially through TGF-β and/or wnt-dependent EMT mechanisms. Citation Format: Ahmed Bakheet, Wen Gao, Dan Cai, Nathan Berger, William Tse, Xiaonan Han. Depletion of Apc gene in Cdx2 gastro-intestinal cells facilitates metastasis of colorectal adenocarcinoma Lgr5 cells to distant organs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1233.

Abstract Background: Caudal-type homeobox 2 (CDX2) is a transcription factor expressed in the gastro-intestinal (GI) epithelial (IEC) and stromal cells. CDX2 along with APC can regulate Lgr5 intestinal stem cell (ISC) differentiation to control GI development or neoplasia. CDX2 or APC loss function is associated with colorectal cancer (CRC) advance. However, whether APC directly regulates CRC metastasis is not clear. We aim to determine the role of inactive Apc gene in Cdx2 GI cells on Lgr5 ISC metastasis. Methods: Tissue sections were collected from colorectal adenocarcinoma or CRC hepatic-metastatic patients, CDX2, CTNNB1, α-SMA, APC and LGR5 expressions were determined. Apc flox mice were crossed with Lgr5CreER and/or Cdx2CreER mice and Apc gene in the Lgr5 ISCs, Cdx2 IECs or stromal cells was inactivated. Cdx2CreER;Apc mice were then crossed with tgfr2 flox mice, tgfr2 was depleted in the Apc-deficient Cdx2 cells. GI tract and liver histology was evaluated and the expression of Lgr5, APC, α-SMA, β-catenin, and CDX2 were examined. The colorectal polyps induced by Apc inactivation were dissected, total RNA was extracted to perform RNA-Seq and quantitative PCR (qPCR) analyses. The colorectal organoids were differentiated. These differentiated organoids were then under intra-abdominal xenotransplantation. Meanwhile, HT-29 cells were transfected with APC and/or CDX2 gRNAs. Results: CTNNB1 and LGR5 expression are increased in human colorectal adenocarcinoma and hepatic-metastatic CRC while interstitial CDX2 and SMA colocalization are robustly pronounced compared to normal colorectum. Induction of Apc depletion in Lgr5 cells in 6-week-old mice leads to Lgr5+ rectal adenocarcinoma and Lgr5 crypts in liver while depletion of Apc in Lgr5 cells in 3-month-old mice results in only Lgr5 intestinal adenoma. 10 day-induction of Apc depletion in Cdx2 cells leads to rectal fibroma and intestinal adenoma as well as undifferentiated metastatic Lgr5 cells in liver. Notably, Apc depletion led to decreased crypt CDX2 while increased colocalization of CDX2 and α-SMA in the stromal cells of adenocarcinoma, and increased expression of TGFBβr2, β-catenin, and EMT markers. Induction of Apc depletion in both Cdx2 and Lgr5 cells led to signet ring cell carcinomas in colorectum and liver. Finally, inducing tgfr2 depletion in Apc-inactive Cdx2 cells impaired GI cancer cell metastasis to liver. Colorectal orgnoids exhibited cancer phenotypes upon Apc depletion in CDX2 or Lgr5 cells. Surprisingly, intra-abdominal xenotransplantation of colorectal organoids exhibited that organoids with Apc deficiency in Lgr5 cells appeared no colonization on or invasion of liver tissues whereas the organoids with Apc deficiency in Cdx2 cells invaded liver. Conclusion: Reduced Apc in Cdx2 cells are required for Lgr5 cell transformation to Lgr5- cancer stem cells. Inactive intestinal Apc increases Cdx2-α-SMA stromal niche cells and facilitates Lgr5 ISC metastasis to liver partially through TGF-β and/or wnt-dependent epithelial-mesenchymal transition (EMT) mechanisms. Citation Format: Xiaonan Han, Ahmed Bakheet, Wen Gao. Depletion of Apc gene in Cdx2 gastro-intestinal cells facilitates metastasis of colorectal adenocarcinoma Lgr5 cells to distant organs [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr A033.

Background: Caudal-type homeobox 2 (CDX2) is a transcription factor expressed in the gastro-intestinal (GI) epithelial (IEC) and stromal cells. CDX2 along with APC can regulate Lgr5 intestinal stem cell (ISC) differentiation to control GI development or neoplasia. CDX2 or APC loss function is associated with colorectal cancer (CRC) advance. However, whether APC directly regulates CRC metastasis is not clear. We aim to determine the role of inactive Apc gene in Cdx2 GI cells on Lgr5 ISC metastasis. Methods: Tissue sections were collected from colorectal adenocarcinoma or CRC hepatic-metastatic patients, CDX2, CTNNB1, α-SMA, APC and LGR5 expressions were determined. Apc flox mice were crossed with Lgr5CreER and/or Cdx2CreER mice and Apc gene in the Lgr5 ISCs, Cdx2 IECs or stromal cells was inactivated. Cdx2CreER;Apc mice were then crossed with tgfr2 flox mice, tgfr2 was depleted in the Apc-deficient Cdx2 cells. GI tract and liver histology was evaluated and the expression of Lgr5, APC, α-SMA, β-catenin, and CDX2 were examined. The colorectal polyps induced by Apc inactivation were dissected, total RNA was extracted to perform RNA-Seq and quantitative PCR (qPCR) analyses. The colorectal organoids were differentiated. These differentiated organoids were then under intra-abdominal xenotransplantation. Meanwhile, HT-29 cells were transfected with APC and/or CDX2 gRNAs. Results: CTNNB1 and LGR5 expression are increased in human colorectal adenocarcinoma and hepatic-metastatic CRC while interstitial CDX2 and SMA colocalization are robustly pronounced compared to normal colorectum. Induction of Apc depletion in Lgr5 cells in 6-week-old mice leads to Lgr5+ rectal adenocarcinoma and Lgr5 crypts in liver while depletion of Apc in Lgr5 cells in 3-month-old mice results in only Lgr5 intestinal adenoma. 10 day-induction of Apc depletion in Cdx2 cells leads to rectal fibroma and intestinal adenoma as well as undifferentiated metastatic Lgr5 cells in liver. Notably, Apc depletion led to decreased crypt CDX2 while increased colocalization of CDX2 and α-SMA in the stromal cells of adenocarcinoma, and increased expression of TGFBβr2, β-catenin, and EMT markers. Induction of Apc depletion in both Cdx2 and Lgr5 cells led to signet ring cell carcinomas in colorectum and liver. Finally, inducing tgfr2 depletion in Apc-inactive Cdx2 cells impaired GI cancer cell metastasis to liver. Colorectal orgnoids exhibited cancer phenotypes upon Apc depletion in CDX2 or Lgr5 cells. Surprisingly, intra-abdominal xenotransplantation of colorectal organoids exhibited that organoids with Apc deficiency in Lgr5 cells appeared no colonization on or invasion of liver tissues whereas the organoids with Apc deficiency in Cdx2 cells invaded liver. Conclusion: Reduced Apc in Cdx2 cells are required for Lgr5 cell transformation to Lgr5- cancer stem cells. Inactive intestinal Apc increases Cdx2-α-SMA stromal niche cells and facilitates Lgr5 ISC metastasis to liver partially through TGF-β and/or wnt-dependent epithelial-mesenchymal transition (EMT) mechanisms. Citation Format: Xiaonan Han, Ahmed Bakheet, Wen Gao. Depletion of Apc gene in Cdx2 gastro-intestinal cells facilitates metastasis of colorectal adenocarcinoma Lgr5 cells to distant organs [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr A033.

Growth hormone insensitivity (GHI) syndrome, first described in 1966, is classically associated with monogenic defects in the GH receptor (GHR) gene which result in severe post-natal growth failure as consequences of insulin-like growth factor I (IGF-I) deficiency. Over the years, recognition of other monogenic defects downstream of GHR has greatly expanded understanding of primary causes of GHI and growth retardation, with either IGF-I deficiency or IGF-I insensitivity as clinical outcomes. Mutations inIGF1and signaling componentSTAT5Bdisrupt IGF-I production, while defects inIGFALSandPAPPA2, disrupt transport and release of circulating IGF-I, respectively, affecting bioavailability of the growth-promoting IGF-I. Defects inIGF1R, cognate cell-surface receptor for IGF-I, disrupt not only IGF-I actions, but actions of the related IGF-II peptides. The importance of IGF-II for normal developmental growth is emphasized with recent identification of defects in the maternally imprintedIGF2gene. Current application of next-generation genomic sequencing has expedited the pace of identifying new molecular defects in known genes or in new genes, thereby expanding the spectrum of GH and IGF insensitivity. This review discusses insights gained and future directions from patient-based molecular and functional studies.