Research

Amit Lab in Action

 

Our research focus is split evenly between experimental cancer cell biology and studying tumor microenvironment subpopulations using computational biology approaches. We develop new experimental methods to isolate and sequence neural niche subpopulations and apply analytical approaches to study how solid tumors sculpt their microenvironment. We focus mainly on head and neck cancer to understand the role of the peripheral nervous system in the evolution of invasion, metastasis and response to chemotherapy. Our goal is to understand the role of neural signaling in tumor evolution so that we can exploit these signals for therapeutic vulnerabilities and enhance cancer therapy. We fully expect that applying these tools to patients will ultimately inform key areas of cancer research including the prevention and treatment of head and neck cancer.

Experimental Models used for Neural-Cancer Cell Interaction

One main reason that understanding mechanisms of neural tracking is challenging is the lack of reliable and reproducible experimental models for neural invasion. The in vitro dorsal root ganglia (DRG) assay (figure part a), designed for studying the neurotropic ability of cancer cells, also enables modulation of paracrine signaling by controlling chemoattractants and by signaling between cancer cells and the DRG. Neurite outgrowth, directionality and Euclidean velocity of cancer cells can be measured before cancer–neuron contact has been established. However, the importance of various cellular components in the classical perineural niche, including Schwann cells and fibroblasts, cannot be directly addressed in this model. 

The recently presented 3D Schwann cell outgrowth and migration assay (figure part b) assesses directional outgrowth of Schwann cells by means of an ex vivo sciatic nerve from newborn models cultured in an extracellular matrix gel suspension. This model enables the early phases of axonogenesis and mutual influences of Schwann cells and cancer cells to be studied.

A novel neural invasion assay (figure part c) enables co-culture of murine fibroblasts with Schwann cells on either side of an 8 μm Millipore cell culture insert membrane with a gelatine separation layer. After 72 hours, labelled cancer cells (with green fluorescent protein (GFP), for example) are added on top to migrate and invade for an additional 24 hours. This assay enables the control and study of the reciprocal interactions between cancer cells, fibroblasts and Schwann cells.

Comparing neurotropic and non-neurotropic cells enables the identification of early genetic and metabolic cellular effects that occur in these cells. An ex vivo perineural invasion model (figure part d) enables the selection of neurotropic subclones using resected rat vagal nerves.

The chicken embryo chorioallantoic membrane (CAM)–DRG model (not shown in the figure), which is used primarily in developmental biology research, includes a rat DRG grafted onto the chicken embryo, which becomes incorporated into connective tissue. Next, cancer cells are inoculated near the DRG; subsequently, neural outgrowth is assessed. This was recently proposed as an in vivo model to address neural outgrowth at tumor inception before cancer cell invasion.

 

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

Solid tumors disseminate in three main ways: direct invasion, lymphatic spread, and hematogenic spread. However, there is a fourth means of cancer spread that is frequently disregarded: dissemination along nerves. This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma. Learn more.

Evolution of Malignant Precursor Lesion that Promotes Cancer Cell Invasion into the Nerve

As a malignant precursor lesion evolves, the perineural niche starts to assemble to form a cellular and biochemical microenvironment that can eventually promote cancer cell invasion into the nerve (panel a). At a certain point, these neurogenic cues initiate axonogenesis, which is accompanied by recruitment of stromal cells typical of the perineural niche (panel b). Subsequent malignant transformation results in the release of multiple chemotactic cues such as stroma-derived C-X-C motif chemokine ligand 13 (CXCL13), which promotes further recruitment of inflammatory cells to establish the perineural niche (panel c). Within the niche, an injured nerve serves as a portal for invasion while neural homeostasis including Wallerian degeneration and nerve regeneration is maintained (panel d). At the perineural niche, the injured nerve maintains regeneration via nerve growth factor (NGF) and neurturin (NRTN) stimulation. Axons secrete C-C motif chemokine ligand 2 (CCL2) that further nourishes the inflammatory response and leads to endoneurial macrophage recruitment. Macrophages secrete glial cell derived neurotrophic factor (GDNF), which facilitates neural tracking and cancer cell invasion by activating RET– GDNF receptor α1 (GFRα1) in the cancer cells. Neuron–tumor crosstalk at the niche is further supported by neurally derived C-X3-C motif chemokine ligand 1 (CX3CL1), which enhances cancer cell adhesion to the nerve, and neurotrophin 3 (NT3), which modulates Schwann cell and cancer cell interaction.

Cancer cell invasion along the neurons of the dorsal root ganglia (DRG)

a. DRG (up) and cancer cells (bottom) on day 0 after seeding. b. DRG and cancer cells on day 7 after seeding. c. DRG extracted from GFP model and cancer cells. d. cancer cells migrate along the DRG neuron (arrows).

Dorsal Root Ganglia: In-Vitro Neural Invasion Modeling System Workflow