STE-01(S)

3.4.1 E xperim ental plan

• Obtain bone marrow aspirates from patients with relapsed prostate cancer and metastatic disease.

• Separate cancer cells from the bone marrow cells.

• Culture prostate cancer cells obtained from the bone marrow samples. • Transduce the prostate cancer cells with PA/tsA58-U19/8.

• Select and expand clones.

3.4.2 R esults

Bone marrow aspirates were obtained from 9 patients with positive bone scans associated with prostate cancer. The bone marrow smears were assessed by a haematologist (Table 3.3).

Sam ple D escription o f sm ear Presence of cancer cells

1 Aparticulate, haemodilute -

2 Aparticulate, normal haemopoietic cells -

3 Cellular aspirate, active haemopoiesis -

4 Haemodilute, fatty -

5 Cellular +

6 Normocellular marrow -

7 Dry tap marrow with few haemopoietic cells +

8 Haemodilute marrow, little normal haemopoietic particles

+

9 No haematological cells seen -

T able 3.3: Cytological analysis of bone m arrow sm ears.

Summary of the report provided by the haematologist on the bone marrow smears taken prior to culture.

E ffect o f lysis buffer on cells derived from the bone marrow

As the majority of the cells in the bone marrow aspirate are red blood cells, removing these cells prior to culture will ensure that the remaining cells (which include the mononuclear

mononuclear white blood cells, bone marrow stromal cells and any circulating cancer cells) can be plated at a higher density. To determine whether the lysis buffer used to rem ove most o f the red blood cells has any adverse effect on the remaining cells in the bone marrow, cells from one sample were spirt and processed with either erythrocyte lysis buffer or WAJC as described in Method 1 (see section 2.2b). The cells were then plated in WAJC supplemented with 1% PCS. The culture treated with the lysis buffer still contained som e red blood cells, but most o f these and the white blood cells were rem oved during subsequent m edia changes, leaving a few colonies o f bone marrow stromal cells. The cells processed with WA.1C contained many red blood cells (resulting in the m edium turning red). Subsequent m edia changes failed to remove a layer o f ‘shrunken’ red blood cells, and no stromal cells grew in this culture.

Growth o f cells derived from the bone marrow

O f the 9 bone marrow samples, 8 were exposed to ammonium chloride lysis buffer to remove the red blood cells. In no case were epithelioid cells seen. B one marrow stromal cells grew in 4 o f these cultures (Table 3.4). The tenth sample was processed with Ficoll to separate the mononuclear cells. Stromal cells also grew in this culture.

The bone marrow stromal cells were between three and five tim es larger than the white blood cells and were polygonal in shape. H owever, w hile proliferating they becom e elongated and grew in whirls characteristic o f fibroblasts in culture (Figure 3.1 ).

Figure 3,1; Culture of stromal cells derived from the bone marrow.

Nucleated cells were plated in the bone marrow medium (B M M ) after separation from the red blood cells.

Sam ple M ethod of separation

M edium used

C ancer cells Cell grow th

Epithelial Strom al cells

1 1 WAJC Absent No No

2 1 WAJC Absent Pungal contamination

3 1 WAJC Absent No No

4 1 WAJC Absent No Yes

5 1+/- Lysis

buffer

WAJC+1% PCS

Present No Only in lysis buffer

treated flask 6 1 WAJC+20% PCS Absent No Yes 7 1 WAJC +/- 1%PCS Present No No 8 1 WAJC+1% PCS/BMM Present No Only in BMM 9 2 BMM/ PrEGM Absent No Only in BMM

T able 3.4: Sum m ary of the results of culture of bone m arrow derived cells +/-: Cells split into two and processed as described.

Method of separation: Method used to separate the nucleated cells from the bone marrow. In method 1, the ammonium chloride lysis buffer was used to remove red blood cells and concentrate the nucleated cells while in method 2 centrifugation through Ficoll was used to concentrate the nucleated cells.

BMM = Bone marrow medium (see section 2.2b).

Cancer cells: As detected by the haematologist from the bone marrow smears taken initially. Cell growth: Cells grown from the aspirate after processing by either method 1 or 2.

There was no growth of bone marrow derived cells (stromal cells or circulating epithelioid cells as determined morphologically) in two out of three cultures plated in serum-free WAJC. Stromal cells (thin, elongated and forming swirls) grew in the remaining culture. To determine whether the inclusion of a high concentration of serum in the medium supports the growth of bone marrow derived cells, 20% serum was included in WAJC to establish one culture. Stromal cells in this culture became confluent within three weeks. To test the effect of a low concentration of serum, cells from one sample were grown in both serum free and serum containing (1%) WAJC. There was no cell growth in either culture.

Pantel and colleagues (Pantel et al, 1995) have used RPMI supplemented with 10% PCS and growth factors (called BMM) to grow bone marrow derived cells and prostate cancer cells. Two samples were split and grown in either WAJC supplemented with 1% PCS or BMM. Stromal cells grew in BMM only. Single cells were seen within three days. To determine whether the new prostate epithelial growth medium PrEGM is suitable for preferentially growing prostate cancer cells from other bone marrow cells, cells from one sample were plated in both BMM or PrEGM in flasks coated with extracellular matrix, after being separated through Picoll. Only the stromal cells in BMM grew.

Immunocytochemical analysis o f bone marrow smears

To determine whether the initial bone marrow aspirate used to set up the cultures contained metastatic prostate cancer cells, smears of each sample were made as described in section 2.2b. Immunocytochemical analysis was carried out on these fixed smears using an pan cytokeratin antibody (detects a mixture of cytokeratin indicating presence of epithelial cells). The smears which contained unusual cells according to the haematologist, were then incubated with anti-PSA antibody to detect the expression of PSA. Of the samples tested, I only detected cytokeratin positive, PSA negative cells in sample 9 in contrast to the three samples in the haematology report.