Cancer Models and Experimental Methods (Lecture 16 and 17)

In-Vitro

Cancer cell lines

• Cancer tissues

• Some lines don’t represent tumor microenvironment

Cancer cells have loss of

E-Cadherin

Loss of E-cadherin can lead too

1. B-catenin to nucleus to transcription

Or

2. Overexpression of growth factors

HIPP-YAP Pathway

Linked to contact-inhibited growth and contains serine/threonine kinases

YAP Normal Conditions

YAP is phosphorylated, no translocation to nucleus and remains cytoplasmic

YAP (loss of E-cadherin or increase in growth factors)

YAP is not phosphorylated and translocates to nucleus

Dependent- Normal

Density and anchorage dependent

• Cells require attachment to ECM to survive, grow and divide

• If lose contact cell goes through anoikis

Dependent - Cancer

Indepedent anchorage and density

• Loss of anoikis (can stay alive)

Anoikis

Programmed cell death when cells lose their attachment to ECM

Unbound Integrins

Trigger apoptosis by prgramming cells to go through IMD (integrin mediated death)

In-Vitro Migration Assay/Boyden Chamber

• Cells added to top chamber

• media conrains GF and attractant/inhibitants

• Cells stained and quantified

What are these Assay cancer cells treated with

RNAi [ShmiR1 + R ; ShmiR2 + R ]

In-Vitro mimic In-vivo

Creates in-vitro environment similar to in-vivo environment

• Addition of cells to matrigel [ECM + GFs]

In-Vitro Angiogenesis Assay [HUVEC]

• Add VEGF and angiogenesis inhbitors (TSP)

• Taxotere and Taxol

Chick Egg CAM Assay

• Placed on chorioallantoic membrane

• Monitored for changes in vascularization

Animal Metastasis Models

• From primary tumor

• Spontaneous and Tail Vein

Spontaneous

Inject cancer cells in animal to look for metastasis

Tail vein

Cancer cells in bloodstream

In-vivo tumors lead to

Tumor spheriods

Tumor Explant

Matric tumor microenvironment

Tumor on a chip

Microfluid system

Spheroid

Organization of cancer cells

Animal Models: Cell proliferation

Soft agar using human breast cancer cells

What are cell proliferation animal models used to treat

Treated to overexpress SIN1

Animal Models: Cell Invasion/Migration

• SIN1 makes for more migration

• See SIN expression and tumor growth

How do you inhibit SIN1

Use shRNA and decreases tumor volume

High SIN1 means

Decrease survival

Molecular Analysis

• Epigenomics

• genomic (DNA)

• transcriptomic (RNA)

• Proteodomes (proteins)

• Metabolism (Warburg effect)

Functional Analysis

• Proliferation

• Apoptosis

• Migration

• Overexpression

Mouse Models: Subc

Inject tumor cells under skin

Mouse Models: Orthotopic

Cells in tissue source

Mouse Models: Syngeneic

Inject mice tumor cells in mice which tumor originated

Clinical Significance: Fresh

Cancer cells and other stromal cells

Clincal Significance: Frozen

Molecular analysis and immunohistochemistry

Epidemilogical Studies

• Suspected carcinogens correlate with human cancers

• Clincal trials: safety and efficacy

Cell Source

• Frozen cancer cell lines from humans

• Examined on petri dish

• Grow as spheroids in a matrix

Microenvironment

Add in ECM (matrigel) and other stromal cells [macrophages and fibroblasts]

Biopsy Samples

Fresh and can use in all models, use activated stroma