Ductal Tree Research Articles

A Single-Cell Atlas of the Murine Pancreatic Ductal Tree Identifies Novel Cell Populations With Potential Implications in Pancreas Regeneration and Exocrine Pathogenesis

Pancreatic ducts form an intricate network of tubules that secrete bicarbonate and drive acinar secretions into the duodenum. This network is formed by centroacinar cells; terminal, intercalated, intracalated ducts; and the main pancreatic duct. Ductal heterogeneity at the single-cell level has been poorly characterized; therefore, our understanding of the role of ductal cells in pancreas regeneration and exocrine pathogenesis has been hampered by the limited knowledge and unexplained diversity within the ductal network. We used small conditional RNA sequencing to comprehensively characterize mouse ductal heterogeneity at single-cell resolution of the entire ductal epithelium from centroacinar cells to the main duct. Moreover, we used organoid cultures, injury models, and pancreatic tumor samples to interrogate the role of novel ductal populations in pancreas regeneration and exocrine pathogenesis. We have identified the coexistence of 15 ductal populations within the healthy pancreas and characterized their organoid formation capacity and endocrine differentiation potential. Cluster isolation and subsequent culturing let us identify ductal cell populations with high organoid formation capacity and endocrine and exocrine differentiation potential invitro, including a Wnt-responsive population, a ciliated population, and FLRT3+ cells. Moreover, we have characterized the location of these novel ductal populations in healthy pancreas, chronic pancreatitis, and tumor samples. The expression of WNT-responsive, interferon-responsive, and epithelial-to-mesenchymal transition population markers increases in chronic pancreatitis and tumor samples. In light of our discovery of previously unidentified ductal populations, we unmask potential roles of specific ductal populations in pancreas regeneration and exocrine pathogenesis. Thus, novel lineage-tracing models are needed to investigate ductal-specific populations invivo.

Decreasing Albumin mRNA Expression in Cholangiocarcinomas along the Bile Duct Tree

Introduction: The progressive technologies in albumin in situ hybridization (ISH) changed the routine application and the differential diagnosis of hepatic malignancies in the last years. The aim of the present work was to assess the diagnostic utility of albumin ISH on different cholangiocarcinoma (CCA) subtypes, as well as to assess how albumin production changes along the biliary tree. Methods: Forty-five CCAs were retrospectively selected: 29 intrahepatic (15 small-duct and 14 large-duct subtypes), 7 perihilar, and 9 extrahepatic. Histology was revised in all cases, and albumin ISH was automatically performed by the RNAscope®. Results: ISH was always negative in extrahepatic CCAs, only 1 perihilar case was positive, and any positivity was observed in 25/29 (86.2%) intrahepatic CCAs (p < 0.001). Concerning CCA subtypes, mean cell positivity was 38.8 ± 29.8% in small-duct CCAs and 11.4 ± 21.9 in large-duct CCAs, respectively (p = 0.003); 12/15 (80.0%) small-duct and 3/14 (21.4%) large-duct CCAs showed >5% positive cells (p = 0.002; odds ratio 14.7). Conclusions: The introduction of more sensitive techniques changed the indications for ISH since most small-duct intrahepatic CCAs show diffuse positivity. Albumin positivity decreases from liver periphery to the large ducts, suggesting that ISH can be helpful in the differential diagnosis between small-duct and large-duct CCAs, as well as between intrahepatic large-duct CCAs and metastases.

Abstract 3389: Intraductal ablative procedure for local preventive intervention in rat models of breast cancer

Abstract There are limited options for primary prevention of breast carcinoma (BC). Prophylactic mastectomy and systemic hormonal therapy are only recommended for high-risk individuals. Prophylactic mastectomy is a drastic surgery that removes the entire breast, including the epithelial cell-lined ductal trees where BC arises but also uninvolved stroma. Even with a 90% risk reduction, many women are deterred from prophylactic mastectomy due to its associated risk of infection, lymphedema, painful recovery time, and mental health toll. Hormonal therapies, tamoxifen and raloxifene, reduce BC risk by upwards of 50% but also increase the risk of uterine cancer and stroke. Thus, new interventions to reduce the risk of BC and minimize deterrents are necessary. Intraductal (ID) injection is a minimally invasive procedure that avoids systemic toxicity by cannulating a needle directly into the ductal tree opening to directly administer a solution to the epithelial cells. We previously reported that ID infusion of 70% ethanol (EtOH) as a cell-killing solution prevents tumor formation in an aggressive genetically engineered mouse model of BC with minimum collateral tissue damage. Here, we investigated the therapeutic efficacy of EtOH-based ablative solutions in a Methyl-N-Nitrosourea (MNU) chemically induced rat model of BC to assess scalability of this procedure towards clinical trials. Two weeks after tumor induction, rats received ID infusions of a solution containing 70% EtOH to ablate epithelial cells, ethyl cellulose (EC) to minimize collateral tissue damage, and/or Tantalum Oxide (TaOx) to visualize infused ductal trees via micro-CT imaging. EtOH-infused glands had the longest tumor latency (273 days vs 97 days in control group, p &amp;lt; 0.0001). No statistically significant differences were noted in epithelial ablation or collateral tissue damage between treatment groups receiving ID injections of 70% EtOH with varying concentrations of EC, but there was a trend to lower collateral damage with EC-containing solutions. Local clearance of TaOx in infused mammary glands enabled post-procedure MR imaging surveillance after 15 d of ID procedure. This preclinical study demonstrates that ID injection of a refined solution containing 70% EtOH and TaOx is an efficacious and scalable procedure for primary prevention of BC. Citation Format: Erin Zaluzec, Mohamed Ashry, Elizabeth G. Phelps, Katarzyna Kempinska, Elizabeth Kenyon, Legend Kenny, Max Volk, Katherine Powell, Jeremy ML Hix, Christiane Mallett, Matti Kiupel, Erik M. Shapiro, Lorenzo F. Sempere. Intraductal ablative procedure for local preventive intervention in rat models of 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 3389.

Mesenchyme instructs growth while epithelium directs branching in the mouse mammary gland.

The mammary gland is a unique organ that undergoes dynamic alterations throughout a female's reproductive life, making it an ideal model for developmental, stem cell and cancer biology research. Mammary gland development begins in utero and proceeds via a quiescent bud stage before the initial outgrowth and subsequent branching morphogenesis. How mammary epithelial cells transit from quiescence to an actively proliferating and branching tissue during embryogenesis and, importantly, how the branch pattern is determined remain largely unknown. Here, we provide evidence indicating that epithelial cell proliferation and onset of branching are independent processes, yet partially coordinated by the Eda signaling pathway. Through heterotypic and heterochronic epithelial-mesenchymal recombination experiments between mouse mammary and salivary gland tissues and ex vivo live imaging, we demonstrate that unlike previously concluded, the mode of branching is an intrinsic property of the mammary epithelium whereas the pace of growth and the density of ductal tree are determined by the mesenchyme. Transcriptomic profiling and ex vivo and in vivo functional studies in mice disclose that mesenchymal Wnt/ß-catenin signaling, and in particular IGF-1 downstream of it critically regulate mammary gland growth. These results underscore the general need to carefully deconstruct the different developmental processes producing branched organs.

Tantalum oxide nanoparticles as versatile and high-resolution X-ray contrast agent for intraductal image-guided ablative procedure in rodent models of breast cancer

AbstractThere are limited options for primary prevention of breast cancer (BC). Experimental procedures to locally prevent BC have shown therapeutic efficacy in animal models. To determine the suitability of FDA-approved iodine-containing and various metal-containing (bismuth, gold, iodine, or tantalum) preclinical nanoparticle-based contrast agents for image-guided intraductal (ID) ablative treatment of BC in rodent models, we performed a prospective longitudinal study to determine the imaging performance, local retention and systemic clearance, safety profile, and compatibility with ablative solution of each contrast agent. At least six abdominal mammary glands (&gt;3 female FVB/JN mice and/or Sprague-Dawley rats, 10–11 weeks of age) were intraductally injected with commercially available contrast agents (Omnipaque® 300, Fenestra® VC, MVivoTM Au, MVivoTM BIS) or in-house synthesized tantalum oxide (TaOx) nanoparticles. Contrast agents were administered at stock concentration or diluted in 70% ethanol (EtOH) and up to 1% ethyl cellulose (EC) as gelling agent to assess their compatibility with our image-guided ablative procedure. Mammary glands were serially imaged by microCT for up to 60 days after ID delivery. Imaging data were analyzed by radiologists and deep learning to measure in vivo signal disappearance of contrast agents. Mammary glands and major organs were ultimately collected for histopathological examination. TaOx-containing solutions provided best imaging performance for nitid visualization of ductal tree immediately after infusion, low outward diffusion (&lt;1 day) and high homogeneity. Of all nanoparticles, TaOx had the highest local clearance rate (46% signal decay as stock and 36% as ablative solution 3 days after ID injection) and exhibited low toxicity. TaOx-containing ablative solution with 1% EC caused same percentage of epithelial cell death (88.62% ± 7.70% vs. 76.38% ± 9.99%, p value = 0.089) with similar minimal collateral damage (21.56 ± 5.28% vs. 21.50% ± 7.14%, p value = 0.98) in mouse and rat mammary glands, respectively. In conclusion, TaOx-nanoparticles are a suitable and versatile contrast agent for intraductal imaging and image-guided ablative procedures in rodent models of BC with translational potential to humans.

Pro-inflammatory cytokines stimulate CFTR-dependent anion secretion in pancreatic ductal epithelium.

Loss of CFTR-dependent anion and fluid secretion in the ducts of the exocrine pancreas is thought to contribute to the development of pancreatitis, but little is known about the impact of inflammation on ductal CFTR function. Here we used adult stem cell-derived cell cultures (organoids) obtained from porcine pancreas to evaluate the effects of pro-inflammatory cytokines on CFTR function. Organoids were cultured from porcine pancreas and used to prepare ductal epithelial monolayers. Monolayers were characterized by immunocytochemistry. Epithelial bicarbonate and chloride secretion, and the effect of IL-1β, IL-6, IFN-γ, and TNF-α on CFTR function was assessed by electrophysiology. Immunolocalization of ductal markers, including CFTR, keratin 7, and zonula occludens 1, demonstrated that organoid-derived cells formed a highly polarized epithelium. Stimulation by secretin or VIP triggered CFTR-dependent anion secretion across epithelial monolayers, whereas purinergic receptor stimulation by UTP, elicited CFTR-independent anion secretion. Most of the anion secretory response was attributable to bicarbonate transport. The combination of IL-1β, IL-6, IFN-γ, and TNF-α markedly enhanced CFTR expression and anion secretion across ductal epithelial monolayers, whereas these cytokines had little effect when tested separately. Although TNF-α triggered apoptotic signaling, epithelial barrier function was not significantly affected by cytokine exposure. Pro-inflammatory cytokines enhance CFTR-dependent anion secretion across pancreatic ductal epithelium. We propose that up-regulation of CFTR in the early stages of the inflammatory response, may serve to promote the removal of pathogenic stimuli from the ductal tree, and limit tissue injury.

The Dynamics of Ductal Tree Remodeling Shape Mammary Field Cancerization

The Dynamics of Ductal Tree Remodeling Shape Mammary Field Cancerization

Safety of lactational exposure to venlafaxine on the rat mammary gland development and carcinogenesis in F1 female offspring

The chronic use of selective serotonin reuptake inhibitors or serotonin-norepinephrine reuptake inhibitors (SNRIs) may result in human gynecomastia, mammoplasia, galactorrhea, and elevated breast cancer risk. As antidepressants are frequently used for postpartum depression (PPD) treatment, this study investigated the adverse effects of lactational exposure to venlafaxine (VENL, a selective SNRI) on mammary gland development and carcinogenesis in F1 female offspring. Thus, lactating Wistar rats (F0) received VENL by oral gavage at daily doses of 3.85, 7.7, or 15.4 mg/kg (N = 9, each group) from lactational day (LD 1) until the weaning of the offspring (LD 21). F1 female offspring were euthanized for mammary gland, and ovary histological analyses on the post-natal day (PND) 22 and 30 (1 pup/litter/period, N = 9, each group). At PND 22, other females (2 pups/litter, N = 18, each group) received a single dose of carcinogen N-methyl-N-nitrosourea (MNU, 50 mg/kg) intraperitoneally (i.p.) for tumor susceptibility assay until PND 250. Tumor incidence and latency were recorded and representative tumor samples were collected for histopathology. The results indicate that lactational exposure to VENL did not alter the development of the mammary gland (epithelial ductal tree or the mean number of terminal end buds), or the ovary (weight and primary, secondary, tertiary, and Graafian follicles) in prepubertal F1 female offspring. In addition, VENL exposure did not influence tumor incidence or tumor latency in adult female offspring that received MNU. Thus, the findings of this animal study indicated that lactational VENL exposure, a period similar to human PPD, did not exert an adverse effect on the mammary gland development at the prepubertal phase or on chemically induced mammary tumorigenesis in adult F1 female rats.

Abstract 4247: Impact of 70% ethanol intraductal injections in MNU rat models for breast cancer prevention

Abstract Breast cancer (BC) is the leading cancer diagnosis in women. While many BC treatment options exist, only two methods are approved for prevention. Prophylactic mastectomy is an invasive surgery that removes the breast, thereby removing the targeted epithelial ductal tree cells, where most BC arises. Despite a 90% risk reduction, recovery time, risk of infection, pain management, and mental health toll on women deter them from this surgery. Hormonal preventatives, tamoxifen and raloxifene, reduce BC risk by up to 50%. However, increased risk of uterine cancer and intolerable sides effect deter women from this option. Therefore, new preventative methods are needed to maintain BC risk reduction while minimizing current BC prevention deterrents. Intraductal (ID) injection is a technique that can circumvent systemic toxicity by inserting a needle directly into the ductal tree opening. We study BC prevention using 70% ethanol (EtOH) as a cell killing, ablative solution for ID injections and have shown its effectiveness of ablation in an aggressive genetically engineered mouse model with minimum collateral tissue damage. EtOH is a safe, inexpensive reagent clinically used as a sclerosing and ablative agent. We introduced ethyl cellulose (EC), a gelling agent which further minimized damage outside the ductal tree. To confirm successful ductal tree filling without animal sacrifice, we introduced Tantalum Oxide (TaOx), a nanoparticle-based contrast agent for real time and long-term visualization of ductal tree filling. TaOx showed remarkable local retention, without impeding 70% EtOH ablative effects or EC compared to other contrast agents. Despite our refined ablative solution, ID injection is a difficult technique to perform with limited ability to confirm success of a fully filled ductal tree. To elucidate any impact of partial ductal tree filling, we utilized a Methyl-N-Nitrosourea (MNU) rat model. Rat models improve scalability toward clinical trials and increase ductal tree filling volume and control to study the impact of partial ductal tree filling. Our MNU rat model provides a time frame between carcinogen exposure and tumor formation optimal for prevention studies. Two weeks after intraperitoneal MNU injections, rats received full or half volume (partial) injections, fat pad (partial), or no injection into individual mammary glands. Rats were monitored for tumor formation from 2 weeks post injection until reaching euthanasia criteria. Fully injected glands had the highest tumor latency increase by 1 month (130 days, p &amp;lt; 0.0001) compared to non-injected controls (95 days). Partial and fat pad tumor latency also improved compared to control (122 days, p &amp;lt;0.01 and 110 days, p &amp;lt;0.05 respectively). No iatrogenic effects of EtOH were observed. Here, we show that ID injection of 70% EtOH is a safe, effective, and scalable technique for BC prevention, which can be enhanced with TaOx and EC for visualization and retention. Citation Format: Erin K. Zaluzec, Mohamed Ashry, Elizabeth Kenyon, Elizabeth G. Phelps, Legend Kenney, Katherine Powell, Maximilian Volk, Shatadru Chakravarty, Jeremy M.L. Hix, Matti Kiupel, Erik Shapiro, Lorenzo F. Sempere. Impact of 70% ethanol intraductal injections in MNU rat models for breast cancer prevention. [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 4247.

Histogenesis of Parotid Gland in Human Fetuses

Background: The present study attempted to find out the histological changes of parotid gland in the developing human fetuses. Materials and Methods: Parotid glands from 60 fresh fetuses of gestational weeks ranging from 12weeks to term were studied after staining with Hematoxylene &amp; Eosin, Masson’s Trichome stain, Van Gieson’s stain and Verhoeff’s stains. Results: The gland of the fetus at 12 weeks composed of solid epithelial cords with occasional canalization, surrounded by loose mesenchyme. Canalization of cords was completed at 25 weeks and adult picture attained at 36 weeks. Division of the glandular parenchyma into lobes and lobules by connective tissue septa started at 17 weeks. A well formed capsule was seen around the gland at 20 weeks. A gradual decrease in intra-glandular connective tissue occurred in late gestational age. Variety of cells such as fibroblasts, mesenchymal cells, fibrocytes and lymphocytes were seen. Numerous adipocytes were found within the glandular parenchyma and around the gland. Ductal tree showed gradual differentiation, presence of cilia-like surface projections from epithelia of larger ducts were seen in 14-32 week fetuses. Conclusion: When compared with various authors, some findings of the present study followed the foot-steps of previous workers whereas some were in contrary, the most important difference being the age of full maturation of the glandular architecture. One of the unique finding being the presence of cilia-like surface projections seen in the larger ducts in 14-32 week aged fetuses.

Abstract F1-2: Clonal evolution of DCIS to invasion

Abstract F1-2: Clonal evolution of DCIS to invasion

In Vivo Gene Delivery into Mouse Mammary Epithelial Cells Through Mammary Intraductal Injection.

Mouse mammary glands comprise ductal trees, which are lined by epithelial cells and have one opening at the tip of each nipple. The epithelial cells play a major role in mammary gland function and are the origin of most mammary tumors. Introducing genes of interest into mouse mammary epithelial cells is a critical step in evaluating gene function in epithelial cells and generating mouse mammary tumor models. This goal can be accomplished through the intraductal injection of a viral vector carrying the genes of interest into the mouse mammary ductal tree. The injected virus subsequently infects mammary epithelial cells, bringing in the genes of interest. The viral vector can be lentiviral, retroviral, adenoviral, or adenovirus-associated viral (AAV). This study demonstrates how a gene of interest is delivered into mammary epithelial cells through mouse mammary intraductal injection of a viral vector. A lentivirus carrying GFP is used to show stable expression of a delivered gene, and a retrovirus carrying Erbb2 (HER2/Neu) is used to demonstrate oncogene-induced atypical hyperplastic lesions and mammary tumors.

Contributions of nanotechnology to the intraductal drug delivery for local treatment and prevention of breast cancer

Breast cancer is a major public health problem, affecting millions of people. It is a very heterogeneous disease, with localized and invasive forms, and treatment generally consists of a combination of surgery and radiotherapy followed by administration of estrogen receptor modulators or aromatase inhibitors. Given its heterogeneity, management strategies that take into consideration the type of disease and biological markers and can provide more personalized and local treatment are required. More recently, the intraductal administration (i.e., into the breast ducts) of drugs has attracted significant attention due to its ability of providing drug distribution through the ductal tree in a minimally invasive manner. Although promising, intraductal administration is not trivial, and difficulties in duct identification and cannulation are important challenges to the further development of this route. New drug delivery strategies such as nanostructured systems can help to achieve the full benefits of the route due to the possibility of prolonging tissue retention, improving targeting and selectivity, increasing cytotoxicity and reducing the frequency of administration. This review aims at discussing the potential benefits and challenges of intraductal administration, focusing on the design and use of nanocarriers as innovative and feasible strategies for local breast cancer therapy and prevention.

A Guide Toward Multi-scale and Quantitative Branching Analysis in the Mammary Gland.

The mammary gland consists of a bilayered epithelial structure with an extensively branched morphology. The majority of this epithelial tree is laid down during puberty, during which actively proliferating terminal end buds repeatedly elongate and bifurcate to form the basic structure of the ductal tree. Mammary ducts consist of a basal and luminal cell layer with a multitude of identified sub-lineages within both layers. The understanding of how these different cell lineages are cooperatively driving branching morphogenesis is a problem of crossing multiple scales, as this requires information on the macroscopic branched structure of the gland, as well as data on single-cell dynamics driving the morphogenic program. Here we describe a method to combine genetic lineage tracing with whole-gland branching analysis. Quantitative data on the global organ structure can be used to derive a model for mammary gland branching morphogenesis and provide a backbone on which the dynamics of individual cell lineages can be simulated and compared to lineage-tracing approaches. Eventually, these quantitative models and experiments allow to understand the couplings between the macroscopic shape of the mammary gland and the underlying single-cell dynamics driving branching morphogenesis.

A Second Ductoscopy Procedure in Patients with Recurrent and Persistent Pathological Nipple Discharge.

Most patients suffering from pathological nipple discharge (PND) undergo local surgical procedures because standard radiological imaging often fails to reveal the cause. Ductoscopy is a minimally invasive endoscopic technique that enables direct intraductal visualization and can avoid unnecessary diagnostic surgical procedures. Hence, patients with recurrent or persistent PND after an unsuccessful ductoscopy procedure still undergo unnecessary surgery. This study describes the experience of a second ductoscopy procedure in patients with recurrent or persistent PND without suspicious radiological findings. Patients with recurrent or persistent PND who underwent two ductoscopy procedures between 2010 and 2017 were retrospectively analyzed. The second ductoscopy was performed when the first ductoscopic attempt was unsuccessful due to technical problems. The primary outcome was the number of preventable surgical procedures. A total of 17 patients underwent two ductoscopy procedures. The first ductoscopy showed a polypoid lesion in 10 patients (58.8%), no abnormalities in 3 patients (17.6%), and in 4 patients (23.5%), it was not possible to visualize the ductal tree. Post-procedure, all patients suffered from PND. After two ductoscopic attempts, PND stopped in 10 patients (58.8%), and 7 patients (41.2%) still suffered from PND and were operated on. Pathology of the resection specimens showed no abnormalities in 1 patient, a papilloma in 5 patients, and ductal carcinoma in situ in 1 patient. A second ductoscopy procedure can be considered in the diagnostic work-up of patients suffering from persistent or recurrent PND after an unsuccessful first ductoscopic attempt to avoid unnecessary surgery in about 59% of the cases.

X-Ray Visualization of Intraductal Ethanol-Based Ablative Treatment for Prevention of Breast Cancer in Rat Models

There are still a limited number of primary interventions for prevention of breast cancer. For women at a high risk of developing breast cancer, the most effective intervention is prophylactic mastectomy. This is a drastic surgical procedure in which the mammary epithelial cells that can give rise to breast cancer are completely removed along with the surrounding tissue. The goal of this protocol is to demonstrate the feasibility of a minimally invasive intraductal procedure that could become a new primary intervention for breast cancer prevention. This local procedure would preferentially ablate mammary epithelial cells before they can become malignant. Intraductal methods to deliver solutions directly to these epithelial cells in rodent models of breast cancer have been developed at Michigan State University and elsewhere. The rat mammary gland consists of a single ductal tree that has a simpler and more linear architecture compared to the human breast. However, chemically induced rat models of breast cancer offer valuable tools for proof-of-concept studies of new preventive interventions and scalability from mouse models to humans. Here, a procedure for intraductal delivery of an ethanol-based ablative solution containing tantalum oxide nanoparticles as X-ray contrast agent and ethyl cellulose as gelling agent into the rat mammary ductal tree is described. Delivery of aqueous reagents (e.g., cytotoxic compounds, siRNAs, AdCre) by intraductal injection has been described previously in mouse and rat models. This protocol description emphasizes methodological changes and steps that pertain uniquely to delivering an ablative solution, formulation consideration to minimize local and systemic side effects of the ablative solution, and X-ray imaging for in vivo assessment of ductal tree filling. Fluoroscopy and micro-CT techniques enable to determine the success of ablative solution delivery and the extent of ductal tree filling thanks to compatibility with the tantalum-containing contrast agent.

Abstract PR005: Intraductal administration of a recombinant transferrin receptor-directed immunotoxin clears ductal carcinoma in situ in preclinical mammary in-duct (MIND) models of breast cancer

Abstract Background: For DCIS, a preneoplastic lesion with a 15-50% chance of recurrence or progression, there is a reappraisal of the necessity of aggressive treatment with its attendant toxicities. HB21(Fv)-PE40, a conjugate of an Fv fragment of the human transferrin-receptor (TFR) mAb and a 40 kDa fragment of bacterial Pseudomonas exotoxin (PE), has potent tumor cell killing activity. HB21(Fv)-PE40 binds to abundantly expressed TFR on cancer cells; the molecule is internalized, processed, and trafficked to the cytosol. Inside the cell, PE-toxin catalyzes the transfer of ADP ribose from nicotinamide adenine dinucleotide (NAD) to elongation factor 2 (EF2), inactivates EF2, and leads to an arrest in protein translation and cell death by apoptosis. However, due to its abundant expression in the liver, the recombinant immunotoxin (RIT) is potentially toxic. We hypothesized that RIT administration via the mammary ductal opening at the teat will destroy preneoplasias, both visible and occult, in the entire ductal tree, while systemic effects will be minimal. The intraductal approach could provide a direct, safe, and highly effective treatment for DCIS. Methods: We tested this concept in two models of DCIS using an ER/PR-, HER2+ cell line derived from a DCIS, SUM225-Luc, and in the ER/PR+, HER2- breast cancer cell line, MCF-7-Luc. In both cell lines, the IC50 of the toxin was in the nM range. DCIS developed within one week in the MIND models following i.duc injection of tumor cells in the 4th pair of mammary glands of female NSG mice. On day 7, the tumor-bearing mice received vehicle, TFR mAb alone, or the HB21(Fv)-PE40 conjugate by the i.duc or i.v route three times at weekly intervals and were observed for 1-6 months. Within two weeks of the treatment, IVIS imaging signals were undetectable in the mammary gland treated with RIT i.duc in both the models (P&amp;lt;0.0001). Mammary gland whole mount analysis, histopathology, and immunohistochemistry for Ki67, Ku80, TFR and CD31 showed that i.duc toxin rendered more than 90% of the mice tumor-free. Pharmacokinetics studies were conducted in mice with the RIT administered i.v. or i.duc. RIT was undetectable by TFR-ELISA in the blood of i.duc-treated mice. Conclusions. Overall, we have shown that HB21(Fv)-PE40 has remarkable antitumor activity by i.duc administration in two MIND models of DCIS. RIT was undetectable in blood after i.duc injection by ELISA, suggesting that the toxin remains inside the ductal system during its 30 min half-life. This preclinical study provides a solid foundation for a clinical study. Dose escalation studies of HB21(Fv)-PE40 by the i.duc route could be performed in patients with DCIS and early breast cancer who will undergo mastectomy as part of their treatment regimen. This study design will allow a detailed examination of the local (breast) and systemic effects of i.duc HB21(Fv)-PE40. It will introduce a potent agent for DCIS treatment and cancer interception in women at high risk of developing breast cancer. The contribution of the immune system remains to be explored. Citation Format: Saraswati Sukumar. Intraductal administration of a recombinant transferrin receptor-directed immunotoxin clears ductal carcinoma in situ in preclinical mammary in-duct (MIND) models of breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr PR005.

EIF3H stabilizes CCND1 to promotes intrahepatic cholangiocarcinoma progression via Wnt/β-catenin signaling.

Cholangiocarcinoma (CCA) is a group of tumors that arise along the human biliary duct tree, ranking second in primary hepatic malignancies. Intrahepatic CCA (iCCA) represents about 10%-20% of CCAs. There is an increasing body of evidence suggesting that iCCAs' incidence and mortality have been increasing globally over the past few decades. In this study, we found that the EIF3H expression level in iCCA tissues was significantly increased compared to the adjacent non-cancerous tissues by immunohistochemistry analysis (IHC). A similar tendency of EIF3H mRNA and protein level was confirmed in iCCA cell lines using RT-qPCR and Western blot. EIF3H has been identified as a critical molecule that plays a pro-neoplasmic role in iCCA both in vivo and in vitro, such as proliferation, migration, and anti-apoptosis. Mechanistically, we found that EIF3H knockdown can promote the degradation of CCND1 and the proteolysis of CCND1 is mediated by ubiquitin-proteasome system (UPS). Thus, we come to the conclusion that EIF3H promotes proliferation and migration of iCCAs, inhibiting apoptosis of iCCA cells at the same time by stabilizing the CCND1 protein structure. Our findings provide insights into the mechanism of tumorigenesis role of EIF3H in iCCAs and a potential therapeutic target for iCCA treatment.

Omniparticle Contrast Agent for Multimodal Imaging: Synthesis and Characterization in an Animal Model.

Individual imaging modalities have certain advantages, but each suffers from drawbacks that other modalities may overcome. The goal of this study was to create a novel contrast agent suitable for various imaging modalities that after a single administration can bridge and strengthen the collaboration between the research fields as well as enrich the information obtained from any one modality. The contrast agent platform is based on dextran-coated iron oxide nanoparticles (for MRI and MPI) and synthesized using a modified co-precipitation method, followed by a series of conjugation steps with a fluorophore (for fluorescence and photoacoustic imaging), thyroxine (for CT imaging), and chelators for radioisotope labeling (for PET imaging). The fully conjugated agent was then tested in vitro in cell uptake, viability, and phantom studies and in vivo in a model of intraductal injection and in a tumor model. The agent was synthesized, characterized, and tested in vitro where it showed the ability to produce a signal on MRI/MPI/FL/PA/CT and PET images. Studies in cells showed the expected concentration-dependent uptake of the agent without noticeable toxicity. In vivo studies demonstrated localization of the agent to the ductal tree in mice after intraductal injection with different degrees of resolution, with CT being the best for this particular application. In a model of injected labeled tumor cells, the agent produced a signal with all modalities and showed persistence in tumor cells confirmed by histology. A fully functional omniparticle contrast agent was synthesized and tested in vitro and in vivo in two animal models. Results shown here point to the generation of a potent signal in all modalities tested without detrimental toxicity. Future use of this agent includes its exploration in various models of human disease including image-guided diagnostic and therapeutic applications.

Exocrine and Endocrine Inflammation Increases Cellular Replication in the Pancreatic Duct Compartment in Type 1 Diabetes.

ContextWe recently demonstrated increased cellular proliferation in the pancreatic ductal gland (PDG) compartment of organ donors with type 1 diabetes, suggesting that PDGs may harbor progenitor cells capable of pancreatic regeneration.ObjectiveWe evaluated the impact of diabetes and pancreatic inflammation on PDG and interlobular duct (ILD) cellular proliferation and profiles.MethodsEndocrine hormone expression (insulin, glucagon, somatostatin, pancreatic polypeptide) and proliferating Ki67+ cells were localized within the PDG and ILD compartments by multicolor immunohistochemistry in cross-sections from the head, body, and tail regions of pancreata from those with (n = 31) or without type 1 diabetes (n = 43). Whole-slide scanned images were analyzed using digital pathology.ResultsType 1 diabetes donors with insulitis or histologically identified pancreatitis had increased cellular replication in the ILD and PDG compartments. Interestingly, while cellular proliferation within the pancreatic ductal tree was significantly increased in type 1 diabetes (PDG mean = 3.36%, SEM = 1.06; ILD mean = 2.78%, SEM = 0.97) vs nondiabetes(ND) subjects without pancreatic inflammation (PDG mean = 1.18%, SEM = 0.42; ILD mean = 0.74%, SEM = 0.15, P < 0.05), robust replication was also observed in ND donors with pancreatitis (PDG mean = 3.52%, SEM = 1.33; ILD mean = 2.18%, SEM = 0.54, P < 0.05). Few polyhormonal cells were present in the ILD (type 1 diabetes = 0.04 ± 0.02%; ND = 0.08 ± 0.03%, P = 0.40) or PDG compartment (type 1 diabetes = 0.02 ± 0.01%; ND = 0.08 ± 0.13%, P = 0.63).ConclusionThese data suggest that increased pancreatic ductal cell replication is associated with sustained pancreatic inflammation; however, as replicating cells were hormone-negative, PDGs do not appear to represent a compelling endogenous source of hormone-positive endocrine cells.