Project Title: Determination of an experimental model of neuroendocrine tumor-mediated fibrosis

Faculty Sponsor: Jill Slater

Department: Biology

Telephone:

E-Mail: jislater@umflint.edu

Project Description: 

Summary:  This project represents an effort to understand the pathology of the desmoplastic reaction which often accompanies small intestine neuroendocrine tumors (SI-NETs) which have metastasized.  “Desmoplasia” is the dense growth of fibrotic or connective tissue.  A desmoplastic reaction is secondary to some insult; in the case of SI-NETs, the tumor is the insult. Student investigator will work on developing the investigational model to be used in further studies.

Briefly, our strategy is to culture murine fibroblasts (NIH-3T3 cells obtained from Dr. Todd Leff, WSU Pathology Department) in conditioned medium from a murine SI-NET cell line (STC-1 cells obtained from Dr. Linda Samuelson, University of Michigan).  Student investigator will determine whether secreted substances from these NET subtypes are able to activate the fibroblasts, leading to their differentiation into myofibroblasts.  This will be determined by measuring markers of each population (e.g. vimentin in fibroblasts, and alpha smooth muscle actin, tenascin C, connective tissue growth factor (CTGF) in myofibroblasts) and possibly of extracellular matrix (ECM) components, such as Type I and V collagen.

Should this prove to be an appropriate model to investigate this question, student investigator will probe the effect of Vitamin D3 and/or PPAR agonist supplementation on subsequent markers of fibrosis.  Each of these has been shown to inhibit fibrosis in other model systems.

Specific aim 1:  Determine whether STC-1 neuroendocrine tumor cell secretions can activate NIH-3T3 fibroblasts, causing them to differentiate into myofibroblasts

Methodology: 

  • Grow STC-1 cells in monolayer and spheroids;make conditioned medium from each type of culture
  • Grow NIH/3T3s in normal medium and conditioned media from step 1.
  • Measure vimentin (marker of inactive fibroblast) v. alphasmooth muscle actin (marker of myofibroblast) expression.  This will be done by Western blot and/or RT-qPCR
  • Compare proliferation rate of NIH/3T3s grown in normal v. conditioned media

Specific aim 2:  If NIH-3T3 fibroblasts do differentiate, determine whether treatment with vitamin D3 or PPAR agonists can prevent or reverse this differentiation.

Methodology:

  • Same as above, but treat NIH/3T3 cells with respective reagent prior to culture.  Measure outcomes as above.

Student Tasks & Responsibilities: Prepare for, attend, and present data at weekly lab meetings Search out and read the pertinent scientific literature pertaining to this project Cell culture Cell viability determination (hemocytometer) Western blot and/or RT-qPCR Data collection/analysis Figure generation, including written descriptions

Minimum Student Qualifications: Willingness to learn, available at least one hour/day (M-F) to work in the lab, responsibility, dependability, integrity, stick-to-itiveness