Author Profile

Investigating the Role of Kinase Inhibitors and Chemotherapeutic Drugs on Pancreatic Cancer Cells

Kristen Johnson, University of New Hampshire-Manchester
Location: New Hampshire

Abstract

Pancreatic Cancer is the fourth deadliest cancer primarily due to its lack of visible symptoms, tremendous invasiveness, and, thus, late stage diagnosis. While much research has focused on understanding the major molecular changes underlying the transition from benign pancreatic ductal epithelial cells to aggressive carcinoma, we are still at a stage where there is no effective therapy. Our lab focuses on the study of transcriptional regulators that we believe play a role in the metastatic stage of tumorigenesis. We are working to dissect the mechanism and pathways in which these transcriptional regulators exert their effects. The research performed in this CURE extends our investigation into one of the transcriptional regulators, a forkhead family protein, FOXN2, through testing kinase inhibitors and chemotherapeutic agents that may deferentially affect cell viability based upon a cell's FOXN2 status. This Cell Culture CURE enables students to (1) perform background research on the transcriptional regulator, inhibitors, and chemotherapeutic agents; (2) write a grant-like proposal for their independent project in which they will testing kinase pathway inhibitors (and/or chemotherapeutic drugs) on cell viability (or other chosen assays); (3) perform the research to collect the data (in triplicate); (4) present their research findings in a campus-wide scientific research conference.

Student Goals

  1. Analyze scientific literature to identify a gap to be addressed by hypothesis-driven independent research project.
  2. Carry out hypothesis driven research project using common cell culture techniques.
  3. Communicate about research progress and findings.

Research Goals

  1. Investigate the function of FOXN2 in cancer, such as lung, liver, and breast, and unpublished pancreatic cancer data (K. Johnson lab) and investigate candidate kinase inhibitors and chemotherapeutic agents for testing.
  2. Determine effect of kinase inhibitors and/or chemotherapeutic agent treatment on pancreatic cancer cells with FOXN2 loss of function.

Context

Cell Culture (BIOT 753) is an advanced lab techniques elective course for our biotechnology majors. Microbiology and Genetics are prerequisites, in which students are exposed to basic aseptic lab techniques and the foundations of DNA and its manipulation. BIOT 753 meets twice a week for 90 minutes in the lecture classroom and twice a week for 2 hours in the laboratory over a typical 16 week semester. Many students who enroll in this course will have extensive basic biology background but they will vary in the upper level courses they have completed both in advanced biology and chemistry. The CURE project takes place during the last 1/3 of the semester and can occur during the lab sessions. However, we tend to run it more as a open lab in which students schedule time in the lab according to their project design.

Note: We have recently launched a masters in biotechnology program, and this course is cross-listed at the 800-level for our masters students, with additional requirements.

Target Audience:Major, Upper Division
CURE Duration:Half a term

CURE Design

Our research question focuses on understanding the role of various transcription factors in epithelial to mesenchymal transition during metastasis in pancreatic cancer. To do this, we have made various RNAi knock-down and CRISPR knockout cells lines to study in culture.

The course-based research is centered around engaging students in basic cell culture techniques utilized in research and industry and encouraging them to use this as a foundation to propose their own project within the CURE theme.
The first half of the semester in lab is structured to teach students the basic cell culture techniques and in lecture content is provided to guide students through content that provides foundational information in order for students to prepare to ask questions and propose a project.

Outside of the classroom, my research lab is most interested in the research findings. When possible, the lab is TA'd by one of my research students, who is able to experience first-hand the outcomes of the research.
More formally, the students enrolled in the course present their findings in a college-wide research conference at the end of the semester.

Core Competencies: Analyzing and interpreting data, Asking questions (for science) and defining problems (for engineering), Planning and carrying out investigations, Using mathematics and computational thinking
Nature of Research:Applied Research, Wet Lab/Bench Research

Tasks that Align Student and Research Goals

Research Goals →
Student Goals ↓
Research Goal 1: Investigate the function of FOXN2 in cancer, such as lung, liver, and breast, and unpublished pancreatic cancer data (K. Johnson lab) and investigate candidate kinase inhibitors and chemotherapeutic agents for testing.
Research Goal 2: Determine effect of kinase inhibitors and/or chemotherapeutic agent treatment on pancreatic cancer cells with FOXN2 loss of function.

Student Goal 1: Analyze scientific literature to identify a gap to be addressed by hypothesis-driven independent research project.

1. Perform background research on FOXN2 and write a specific aims page for an NIH-like grant proposal to outline the gap in knowledge and how the project design will address the knowledge gap. 
2. Perform background research to choose kinase inhibitor(s) and/or chemotherapeutic agent(s) to be used in proposed experiments and determine the concentration range(s) to treat the pancreatic cancer cells. This information will inform students' experimental design.

1. Establish the hypothesis that drives the individual experimental design. 
2. Carefully design cell culture assays to assess cell viability after the addition of chosen kinase inhibitor(s) and/or chemotherapeutic agent(s).


Student Goal 2: Carry out hypothesis driven research project using common cell culture techniques.

2. Present a detailed outlined and project plan for the experimental design including technical and biological replicate numbers, materials and methods, and expected outcomes.

1. Carry out experiments as designed and collect data in multiple replicates.
2. Analyze data to determine statistical significance.


Student Goal 3: Communicate about research progress and findings.

1. During the first lab meetings, discuss the background, hypothesis, project proposal and experimental design with the instructor and other students to receive feedback. 
2. Report upon final research findings through a poster accompanied by a oral presentation at the end of semester research conference. The talk includes the research about background and establishment of study objectives and hypotheses.

1. Maintain an online lab notebook throughout the research process to document progress.
2. Meet weekly in a lab meeting with other classmates (and instructor) to discuss project progress, pitfalls, data analysis approaches and problem solving techniques.
3. Final poster accompanied by an oral presentation, including a heavy focus on data and analysis, conclusions and future steps.


Instructional Materials

Attached are files used in this CURE in a zipped folder, including:
1. Project Proposal Assignment: Instructions for introduce the students for performing research about the topic and for the structure for writing the grant specific aims and experimental design sections. Grading Rubric is included at the end of this document.
2. Independent Project Outline: Instructions to assist students with details of experimental design.
3. Cytotoxicity Lab: This is normally performed during the early part of the semester to teach the students the basic principles of a cytotoxicity experiment before they design their own in this CURE. Examples of drug inhibitor dilutions/calculations are included in this protocol.
4. Experimental Set-Up Flow Chart: Powerpoint file for instructor to use to demonstrate the protocols; also good for student reference.
5. Inhibitor Treatment Dilution Chart: Blank chart for students to use to set up inhibitor/chemotherapy treatments for assays.
6. Independent Project Milestones: Timeline/syllabus for this CURE. Note: The weeks preceding this in the lab are used to establish aseptic technique and basic cell culture skills.

Instructional Materials and Student Handouts for Cancer CURE (Zip Archive 2MB Aug17 21)

Assessment

In order to determine whether we have met the goals of a CURE in this portion of the course, we have an IRB for the students to take two surveys: (1) Laboratory Course Assessment Survey (LCAS) (Corwin, 2015) in order to assess whether the elements of the CURE have been achieved through its implementation; (2) Persistence in the Sciences (PITS) survey (Hanahauer, 2016) to assess persistence in STEM.  Our data are included in a larger data collection effort regarding Cell Biology based CUREs with the Cell Biology Education Consortium (CBEC.org).

In order to determine whether students have met the research goals, they are assessed on their final project poster and oral presentation (rubric is attached).  Additionally, their grant proposal and experimental design is assessed (rubric is attached to the proposal assignment in the zipped folder).  Finally, their online lab notebooks are assessed.

CURE Rubric for Independent Project and Poster Presentation (Microsoft Word 2007 (.docx) 32kB Aug17 21)

Instructional Staffing

This CURE is largely run by me as the main instructor. At times, I am able to hire an undergraduate TA who can help with the preparation of media and other supplies for the lab and who is knowledgeable about the techniques and cell lines that the students are working with. Most often this student is from my research lab which has been very helpful.

Author Experience

Kristen Johnson, University of New Hampshire-Manchester

Few undergraduates are able to engage in Cell Culture based research in a faculty lab, particularly at small institutions. This Cell Culture based CURE allows students to conduct such research after gaining basic skills within a course setting.


Advice for Implementation

While this CURE is rather specific to my area of research, the transcription factor that we study, and cell lines that we have made, the basic principles behind it can be applied to other cancer cell lines. 

The most important and successful features of the CURE, I have found, are the research and planning phases that result in the writing of a grant specific aims page. The students really hone their technical writing skills through this process. The careful experimental planning before actually conducting their experiments includes a timeline using a calendar in order for the student to understand how achievable their plan and incorporation of replicates will be. Invariably, there are issues with the implementation, and problem solving and further iterations of the process are carried out. This occurs during the weekly lab meetings and is where much of the real learning takes place.

While I recommend that students work in groups of two for the project, some students choose to work alone. I have found that both scenarios work out just fine in terms of project outcomes.

Iteration

The students are required to plan and carry out three biological replicates within their projects (and many technical replicates within each biological replicate to understand variability). These biological replicates allow them to troubleshoot and problem solve as they may see things go differently than expected or need to repeat even more if the protocol wasn't followed precisely. Students carry out statistical analysis using JMP once data collection is complete.

Using CURE Data

Most students choose different inhibitors and chemotherapeutic combinations to test in their project. However, when there is overlap within the class, students are encouraged to share their data with peer groups and to compare their findings. All data are analyzed statistically for significance using JMP. At this time, no publications have resulted from this CURE. However, all poster presentations and oral presentations at our undergraduate research conference have had all students researchers attributed as authors.

Resources

Assessment References:

  • Corwin, L. A., Runyon, C., Robinson, A., & Dolan, E. L. (2015). The Laboratory Course Assessment Survey: A Tool to Measure Three Dimensions of Research-Course Design. CBE—Life Sciences Education, 14(4), ar37. https://doi.org/10.1187/cbe.15-03-0073
  • Hanauer, D. I., Graham, M. J., & Hatfull, G. F. (2016). A Measure of College Student Persistence in the Sciences (PITS). CBE Life Sciences Education, 15(4). https://doi.org/10.1187/cbe.15-09-0185

Research Resources (appropriate for both instructors and students, and referenced in the instructional material posted):

  • Amano, M., Nakayama, M., & Kaibuchi, K. (2010). Rho-Kinase/ROCK: A Key Regulator of the Cytoskeleton and Cell Polarity. Cytoskeleton (Hoboken, N.j.), 67(9), 545–554. https://doi.org/10.1002/cm.20472
  • Conway, J. R., Herrmann, D., Evans, T. J., Morton, J. P., & Timpson, P. (2019). Combating pancreatic cancer with PI3K pathway inhibitors in the era of personalised medicine. Gut, 68(4), 742–758. https://doi.org/10.1136/gutjnl-2018-316822
  • Morgan-Fisher, M., Wewer, U. M., & Yoneda, A. (2013). Regulation of ROCK Activity in Cancer. Journal of Histochemistry and Cytochemistry, 61(3), 185–198. https://doi.org/10.1369/0022155412470834
  • Santibanez, B., Oliveira, S., Duggan, K., Lu, K., & Johnson, K. (2019). Abstract C49: Knockdown of FOXN2 enhances adhesion and reduces migration in pancreatic cancer cells. Cancer Research, 79(24 Supplement), C49–C49. https://doi.org/10.1158/1538-7445.PANCA19-C49
  • Vennin, C., Chin, V. T., Warren, S. C., Lucas, M. C., Herrmann, D., Magenau, A., Melenec, P., Walters, S. N., Del Monte-Nieto, G., Conway, J. R. W., Nobis, M., Allam, A. H., McCloy, R. A., Currey, N., Pinese, M., Boulghourjian, A., Zaratzian, A., Adam, A. A. S., Heu, C., ... Timpson, P. (2017). Transient tissue priming via ROCK inhibition uncouples pancreatic cancer progression, sensitivity to chemotherapy, and metastasis. Science Translational Medicine, 9(384), eaai8504. https://doi.org/10.1126/scitranslmed.aai8504
  • Vennin, C., Rath, N., Pajic, M., Olson, M. F., & Timpson, P. (2020). Targeting ROCK activity to disrupt and prime pancreatic cancer for chemotherapy. Small GTPases, 11(1), 45–52. https://doi.org/10.1080/21541248.2017.1345712
  • Wang, Z., Luo, G., & Qiu, Z. (2020). Akt inhibitor MK-2206 reduces pancreatic cancer cell viability and increases the efficacy of gemcitabine. Oncology Letters, 19(3), 1999–2004. https://doi.org/10.3892/ol.2020.11300
  • Wei, L., Surma, M., Shi, S., Lambert-Cheatham, N., & Shi, J. (2016). Novel Insights into the Roles of Rho Kinase in Cancer. Archivum Immunologiae et Therapiae Experimentalis, 64, 259–278. https://doi.org/10.1007/s00005-015-0382-6

*Techniques resources:

  • Freshney's Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 8th Edition | Wiley. (n.d.). Wiley.Com. Retrieved August 17, 2021, from https://www.wiley.com/en-gb/Freshney%27s+Culture+of+Animal+Cells%3A+A+Manual+of+Basic+Technique+and+Specialized+Applications%2C+8th+Edition-p-9781119513049 [Chapter 24]
  • Useful site for investigating inhibitor compounds: www.selleckchem.com
  • Presto Blue Protocol: https://www.thermofisher.com/us/en/home/references/protocols/cell-and-tissue-analysis/protocols/prestoblue-cell-viability-reagent-for-microplates-protocol