Self-Renewal Properties of LAM Cells

Personnel: Dr James Baty and Research Professor Mike Berridge

Primary project aims

1. To determine whether LAM cells are capable of growing under conditions that favour the generation of self-renewing cancer stem cell-like cells.
2. To determine whether LAM cells grown under “cancer stem cell” conditions express increased self-renewal genes compared with cells grown in normal conditions.

Summary of key achievements to date

LAM cells from the lungs of three patients and non-LAM lung smooth muscle cells from two individuals are now growing in our laboratory. These cells were collected by Prof Judy Black and Dr Lyn Moir (Woolcock Institute of Medical Research, Sydney) from lung transplant patients and delivered to the Malaghan Institute frozen on dry ice. Some of the growing cells have been re-frozen and are being stored in liquid nitrogen for future experiments. All of the cell types tested have shown expression of smooth muscle actin. However, we have had difficulty observing increased staining of the LAM cells with the antibody HMB45. This could be due to the antibody itself, so new antibody has been ordered from a different supplier and these experiments will be repeated.

We have also recently taken delivery of LAM cells from an additional three patients including cells from lungs cysts, and cells that Dr Moir and Prof Black obtained from chylous fluid from the lungs of a LAM patient. We plan to investigate the stem cell properties of these cells and see if they behave differently from the LAM cells taken from nodules growing in the lungs of patients. (Thanks to LARA for funding the delivery costs).

Serum-free cell growth: Results so far have indicated that all the cell types are able to survive in serum-free culture medium (“stem-cell favouring conditions”). There is some evidence of non-adherent “sphere” formation, which is usually indicative of stem cells. However, while spheres/clusters of cells form initially they do not appear to reform after passaging.

Expression of self-renewal genes: Our experiments have shown that cells growing in serum-free culture medium increase the expression of some genes involved in cellular self-renewal (Sox2, BMPR2, possibly Oct4 and Msi1). Up-regulation of these genes can be indicative of a population of stem cells. Intriguingly, non-LAM lung cells growing under these conditions also showed increased expression of self-renewal genes. One possibility is that these cells represent a population of cells with self-renewal potential in non-LAM lungs.

Hypoxia (low oxygen conditions): LAM and non-LAM cells have been cultured in 1% oxygen to see if they are able to grow under low oxygen conditions. The results so far suggest that they are indeed able to survive and grow however the system needs some refinement to obtain more reliable results.

17- β-estradiol: Experiments investigating the effect of 17- β-estradiol on LAM cell growth over a few days have so far been inconclusive so we will be testing a slightly different experimental approach so determine if 17- β-estradiol is able to stimulate the growth of the LAM cells. All the additional materials required for these experiments have now arrived and so this work will be conducted in the very near future.

Estrogen receptor: It has been reported by others that LAM cells have higher expression of the receptor for estrogen, which would explain their response to this hormone. We have investigated the expression of the estrogen receptor subunits α and β in LAM and non-LAM cells. Our experiments have indicated that the expression of the βsubunit is higher in 2 of 3 LAM cell types when compared to non-LAM smooth muscle cells. We plan to further investigate estrogen receptor expression in the cells by looking at the levels of actual estrogen receptor protein using specific antibodies.

Finally, we were successful with an application to the Wellington Medical Research Foundation for a grant-in-aid of research for working expenses that will supplement our current funding for this project and allow us to explore some additional questions.