Biology subjects

Carlson, M. A.

Publications and source records attributed to Carlson, M. A..

8 recordsLinked to original sources

bioRxiv2024

SELECTIVE EPITHELIAL ACTIVATION OF KRASG12D DRIVES DUCTAL PANCREATIC NEOPLASIA IN SWINE

BackgroundPancreatic ductal adenocarcinoma (PDAC) remains a formidable challenge in oncology, characterized by a high mortality rate, largely attributable to delayed diagnosis and the intricacies of its tumor microenvironment. Innovations in modeling pancreatic epithelial transformation provide valuable insights into the pathogenesis and potential therapeutic strategies for PDAC. MethodsWe employed a porcine (Oncopig) model, utilizing the Ad-K8-Cre adenoviral vector, to investigate the effects of variable doses (107 to 1010 pfu) on pancreatic epithelial cells. This vector, the expression from which being driven by a Keratin-8 promoter, will deliver Cre-recombinase specifically to epithelial cells. Intraductal pancreatic injections in transgenic Oncopigs (LSL-KRASG12D-TP53R167H) were performed with histologically based evaluation at 2 months post-injection. ResultsSpecificity of the adenoviral vector was validated through Keratin-8 expression and Cre-recombinase activity. We confirmed that the Ad-K8-Cre adenoviral vector predominantly targets ductal epithelial cells lining both large and small pancreatic ducts, as evidenced by Keratin 8 and CAM5.2 staining. Higher doses resulted in significant tissue morphology changes, including atrophy, and enlarged lymph nodes. Microscopic examination revealed concentration-dependent proliferation of the ductal epithelium, cellular atypia, metaplasia, and stromal alterations. Transgene expression was confirmed with immunohistochemistry. Desmoplastic responses were evident through vimentin, -SMA, and Massons trichrome staining, indicating progressive collagen deposition, particularly at the higher vector doses. ConclusionOur study suggests a distinct dose-response relationship of Ad-K8-Cre in inducing pancreatic epithelial proliferation and possible neoplasia in an Oncopig model. All doses of the vector induced epithelial proliferation; the higher doses also produced stromal alterations, metaplasia, and possible neoplastic transformation. These findings highlight the potential for site-specific activation of oncogenes in large animal models of epithelial tumors, with the ability to induce stromal alterations reminiscent of human PDAC.

cancer biology↗

bioRxiv2021

Ectopic expression of KRASG12D and p53R167H in porcine mammary epithelial cells results in transformation and tumorigenesis

We describe our initial studies in the development of an orthotopic, genetically-defined, large animal model of breast cancer, using immunocompetent pigs. Primary mammary epithelial cells were isolated from the porcine gland. Primary mammary cells were immortalized with hTERT, and then transformed cell lines were generated from these immortalized cells with oncogenic KRAS and dominant negative p53. The transformed cell lines outperformed the primary cells in terms proliferation, population doubling time, soft agar growth, 2D migration, and Matrigel invasion. Three transformed cell lines were selected based on in vitro performance, and were able to grow tumors when injected subcutaneously in nude mice, with undifferentiated morphology. Tumorigenic porcine mammary cell lines were generated in this report.

cancer biology↗

bioRxiv2021

Isolating and Cryo-Preserving Pig Skin Cells for Single Cell RNA Sequencing Study

The Pigskin architecture and physiology are similar to these of humans. Thus, the pig model is valuable for studying skin biology and testing therapeutics for skin diseases. The single-cell RNA sequencing technology allows quantitatively analyzing cell types, cell states, signaling, and receptor-ligand interactome at single-cell resolution and at high throughput. scRNA-Seq has been used to study mouse and human skins. However, studying pigskin with scRNA-Seq is still rare. Here we described a robust method for isolating and cryo-preserving pig single cells for scRNA-Seq. We showed that pigskin could be efficiently dissociated into single cells with high cell viability using the Miltenyi Human Whole Skin Dissociation kit and the Miltenyi gentleMACS Dissociator. Also, we showed that the subsequent single cells could be cryopreserved using DMSO without causing additional cell death, cell aggregation, or changes in gene expression profiles. Using the developed protocol, we were able to identify all the major skin cell types. The protocol and results from this study will be very valuable for the skin research scientific community.

cell biology↗

bioRxiv2021

Porcine pancreatic ductal epithelial cells transformed with KRASG12D and SV40T are tumorigenic

We describe our initial studies in the development of an orthotopic, genetically-defined, large animal model of pancreatic cancer. Primary pancreatic epithelial cells were isolated from pancreatic duct of domestic pigs. A transformed cell line was generated from these primary cells with oncogenic KRAS and SV40T. The transformed cell lines outperformed the primary and SV40T immortalized cells in terms of proliferation, population doubling time, soft agar growth, transwell migration and invasion. The transformed cell line grew tumors when injected subcutaneously in nude mice, forming glandular structures and staining for epithelial markers. Future work will include implantation studies of these tumorigenic porcine pancreatic cell lines into the pancreas of allogeneic and autologous pigs. The resultant large animal model of pancreatic cancer could be utilized for preclinical research on diagnostic, interventional, and therapeutic technologies.

cancer biology↗

bioRxiv2020

The effect of pre-resection obesity on post-resection body composition after 75% small bowel resection in rats

BackgroundIn patients with short bowel syndrome, an elevated pre-resection Body Mass Index may be protective of post-resection body composition. We hypothesized that rats with diet-induced obesity would lose less lean body mass after undergoing massive small bowel resection compared to non-obese rats. MethodsRats (CD(R)IGS; age = 2 mo; N = 80) were randomly assigned to either a high-fat (obese rats) or a low-fat diet (non-obese rats), and fed ad lib for six months. Each diet group then was randomized to either underwent a 75% distal small bowel resection (massive resection) or small bowel transection with re-anastomosis (sham resection). All rats then were fed ad lib with an intermediate-fat diet (25% of total calories) for two months. Body weight and quantitative magnetic resonance-determined body composition were monitored. ResultsPreoperative body weight was 884 {+/-} 95 vs. 741 {+/-} 75 g, and preoperative percent body fat was 35.8 {+/-} 3.9 vs. 24.9 {+/-} 4.6%; high-fat vs. low fat diet, respectively (p < 0.0001); preoperative diet type had no effect on lean mass. Regarding total body weight, massive resection produced an 18% vs. 5% decrease in high-fat vs. low-fat rats respectively, while sham resection produced a 2% decrease vs. a 7% increase, respectively (p < 0.0001, preoperative vs. necropsy data). Sham resection had no effect on lean mass; after massive resection, both high-fat and low-fat rats lost lean mass, but these changes were not different between the latter two rat groups. ConclusionThe high-fat diet and low-fat diet induced obesity and marginal obesity, respectively. The massive resection produced greater weight loss in high-fat rats compared to low-fat rats. The type of dietary preconditioning had no effect on lean mass loss after massive resection. A protective effect of pre-existing obesity on lean mass after massive intestinal resection was not demonstrated.

physiology↗

bioRxiv2020

Induction of pancreatic neoplasia in the KRAS/TP53 Oncopig: preliminary report

IntroductionFive year survival of pancreatic cancer (PC) remains low. Current murine models may not adequately mimic human PC and can be too small for medical device development. A large animal PC model could address these issues. We induced and characterized pancreatic tumors in Oncopigs (transgenic swine with a somatic floxed cassette containing KRASG12D and TP53R167H). MethodsOncopigs underwent injection of adenovirus expressing Cre recombinase (AdCre) +/- interleukin 8 (IL-8) into one of the main pancreatic ducts (induction procedure). Subjects were necropsied after [≤]10 week, followed by histological analysis, cytokine expression analysis, exome sequencing and transcriptome analysis of resultant tumors. ResultsFourteen Oncopigs underwent the induction procedure; ten (71%) had gross tumor within three weeks, one of these subjects expired suddenly and the other 9 required premature euthanasia secondary to lack of oral intake. At necropsy all of ten of these subjects had gastric outlet obstruction secondary to pancreatic tumor and phlegmon. Two Oncopigs underwent a control injection (no AdCre) and four WT littermates of the Oncopigs underwent AdCre injection without notable effect. Exome and transcriptome analysis of the porcine pancreatic tumors revealed similarity with the molecular signatures and pathways of human PC. ConclusionOncopigs with ductal injection of AdCre developed pancreatic tumor in a short period of time with molecular characteristics similar to human PC. While further optimization and validation of this porcine PC model would be beneficial, it is anticipated that this model will be useful for focused research and development of diagnostic and therapeutic technologies for PC.

cancer biology↗

bioRxiv2020

Three-Dimensional (3D) Fibronectin Nano-Array Presented on Fibrin Matrix Accelerates Mice Skin Wound Healing

Plasma fibrinogen (F1) and fibronectin (pFN) polymerize to form a fibrin clot that is both a hemostatic and provisional matrix for wound healing. About 90% of plasma F1 has a homodimeric pair of {gamma} chains ({gamma}{gamma}F1) and 10% has a heterodimeric pair of {gamma} and more acidic {gamma} chains ({gamma}{gamma}F1). We have synthesized a novel fibrin matrix exclusively from a 1:1 (molar ratio) complex of {gamma}{gamma}F1 and pFN in the presence of highly active thrombin and recombinant Factor XIII (rFXIIIa). In this matrix, the fibrin nanofibers were wrapped with periodic 200-300 nm wide pFN nanobands (termed {gamma}{gamma}F1:pFN fibrin). In contrast, fibrin made from 1:1 mixture of {gamma}{gamma}F1 and pFN formed a sporadic distribution of "pFN droplets" (termed {gamma}{gamma}F1 +pFN fibrin). The {gamma}{gamma}F1:pFN fibrin enhanced the adhesion of primary human umbilical vein endothelium cells (HUVECs) relative to the {gamma}{gamma}F1+FN fibrin. Three dimensional (3D) culturing showed that the {gamma}{gamma}F1:pFN complex fibrin matrix enhanced the proliferation of both HUVECs and primary human fibroblasts. HUVECs in the 3D {gamma}{gamma}F1:pFN fibrin exhibited a starkly enhanced vascular morphogenesis while an apoptotic growth profile was observed in the {gamma}{gamma}F1 +pFN fibrin. Relative to {gamma}{gamma}F1 +pFN fibrin, mouse dermal wounds that were sealed by {gamma}{gamma}F1:pFN fibrin exhibited accelerated and enhanced healing. This study suggests that a 3D pFN nano-array presented on a fibrin matrix can promote wound healing.

bioengineering↗

bioRxiv2018

Collateral development and arteriogenesis in hindlimbs of domestic swine after ligation of arterial inflow

IntroductionThe development of collateral vasculature is a key mechanism compensating for arterial occlusions in patients with peripheral artery disease (PAD). We aimed to examine the development of collateral pathways after ligation of native vessels in a porcine model of PAD. MethodsRight hindlimb Ischemia was induced in domestic swine (N=11, male, kg) using two different versions of arterial ligation. Version 1 (N=6) consisted of ligation/division of the right external iliac, profunda femoral (RPFA) and superficial femoral arteries (RSFA). Version 2 (N=5) consisted of the ligation of Version 1 with additional ligation/division of the right internal iliac artery (RIIA). Development of collateral pathways was evaluated with standard angiography at baseline (prior to arterial ligation) and at termination (4-8 weeks later). Relative luminal diameter of the arteries supplying the ischemic right hindlimb were determined by 2D angiography, as percent of the size of the distal aortic diameter. ResultsThe dominant collateral pathway that developed after version 1 ligation connected the RIIA to the RPFA and RSFA/popliteal artery. Mean luminal diameter ({+/-} standard error) of the RIIA at termination increased by 38% (P<0.05) compared to baseline. Two co-dominant collateral pathways developed in version: (i) from the common internal iliac trunk and left internal iliac artery to the reconstituted RIIA, which then supplied the RPFA and RSFA/popliteal arteries; and (ii) from left profunda artery to the reconstituted RPFA. Mean diameter of the common internal iliac trunk and left profunda artery both increased at termination in the range of 20% (p < 0.05). ConclusionTwo versions of hindlimb ischemia induction (right ilio-femoral artery ligation with and without right internal iliac artery ligation in swine produced differing collateral pathways, along with changes to the diameter of the inflow vessels (i.e., arteriogenesis). Radiographic and anatomical data of the collateral formation in this porcine model should have value in investigation of the pathophysiology of hindlimb ischemia, and assessment of angiogenic therapies as potential treatments for PAD.

physiology↗