3 Methods
In this section, we describe the workflow to assess flies for distinct
steps of metastatic cancer; it is subdivided into four sections. Firstly,
the flies with clones of mutant intestinal cells are generated, control
lines are generated in parallel. Next, the flies are screened for
secondary tumors. Third, the primary tumor burden is analyzed
by either detecting clones visually by GFP or by enzymatic reaction
to detect for luciferase. Finally, the hemolymph of the flies is
screened for circulating tumor cells. In the notes, we will discuss
the advantages and disadvantages of detecting GFP vs luciferase as
we go through each detection assay.
3.1 Generation
of the Flies
for Analysis
1. Cross 30 females and 10 males in each vial according to the
mating schemes depicted in Materials. Collect females that lack
CyO and TMBb balancers.
2. Collect females over a period of 6 days. On the seventh day, add
20 female and 3 male (see Note 1) flies to a vial. Wrap the tops
with parafilm and heat shock for 1 h in a 37
C water bath. The
vials should be totally submerged to ensure that all flies are
subjected to the heat, and spread as equally as possible for a
uniform heat shock (Fig. 1).
3. Flies are placed in fresh vials of food after the heat shock and
flipped every 4 days. This is to ensure that flies are not stuck to
the food.
3.2 Screening Flies
for Secondary
Metastases
1. Screen flies every 3–4 days for metastases on the fluorescence
stereo microscope. Primary tumors in ApcRasSna flies grow in
the midgut and Malpighian tubules (MpTs, the kidneys of the
fly). These appear as small dots or lumps of GFP (Fig. 2a).
Generally, secondary tumors can be distinguished from primary
tumors as they tend to be larger, brighter, and often closer to
the surface than tumors in the intestinal tract (Fig. 2b).
2. Confirm metastasis by dissecting out the gastrointestinal tract.
To do this, first anaesthetize the fly with CO 2 , and place in a
drop of PBS on a microscope slide. Cut the head cleanly off the
fly with a pair of forceps. Next pinch the anus of the fly with one
forceps, and the thorax of the fly between stripes 3 and 4 with
the other (Fig. 3a). Gently pull until the proventriculus, and
the 4 MpTs pop out (Fig. 3b) (see Note 2).
3. Check and see if masses of GFP positive cells separated from the
gastrointestinal tract come out in the hemolymph (Fig. 4a) and
to see if any GFP positive masses are left in the body (Fig. 4b).
These tumors can then be imaged live by mounting in 10 S oil,
or fixed and stained, using standard methods for fixing and
staining adult midguts (see Note 3).
164
Jamie Adams et al.
In this section, we describe the workflow to assess flies for distinct
steps of metastatic cancer; it is subdivided into four sections. Firstly,
the flies with clones of mutant intestinal cells are generated, control
lines are generated in parallel. Next, the flies are screened for
secondary tumors. Third, the primary tumor burden is analyzed
by either detecting clones visually by GFP or by enzymatic reaction
to detect for luciferase. Finally, the hemolymph of the flies is
screened for circulating tumor cells. In the notes, we will discuss
the advantages and disadvantages of detecting GFP vs luciferase as
we go through each detection assay.
3.1 Generation
of the Flies
for Analysis
1. Cross 30 females and 10 males in each vial according to the
mating schemes depicted in Materials. Collect females that lack
CyO and TMBb balancers.
2. Collect females over a period of 6 days. On the seventh day, add
20 female and 3 male (see Note 1) flies to a vial. Wrap the tops
with parafilm and heat shock for 1 h in a 37
C water bath. The
vials should be totally submerged to ensure that all flies are
subjected to the heat, and spread as equally as possible for a
uniform heat shock (Fig. 1).
3. Flies are placed in fresh vials of food after the heat shock and
flipped every 4 days. This is to ensure that flies are not stuck to
the food.
3.2 Screening Flies
for Secondary
Metastases
1. Screen flies every 3–4 days for metastases on the fluorescence
stereo microscope. Primary tumors in ApcRasSna flies grow in
the midgut and Malpighian tubules (MpTs, the kidneys of the
fly). These appear as small dots or lumps of GFP (Fig. 2a).
Generally, secondary tumors can be distinguished from primary
tumors as they tend to be larger, brighter, and often closer to
the surface than tumors in the intestinal tract (Fig. 2b).
2. Confirm metastasis by dissecting out the gastrointestinal tract.
To do this, first anaesthetize the fly with CO 2 , and place in a
drop of PBS on a microscope slide. Cut the head cleanly off the
fly with a pair of forceps. Next pinch the anus of the fly with one
forceps, and the thorax of the fly between stripes 3 and 4 with
the other (Fig. 3a). Gently pull until the proventriculus, and
the 4 MpTs pop out (Fig. 3b) (see Note 2).
3. Check and see if masses of GFP positive cells separated from the
gastrointestinal tract come out in the hemolymph (Fig. 4a) and
to see if any GFP positive masses are left in the body (Fig. 4b).
These tumors can then be imaged live by mounting in 10 S oil,
or fixed and stained, using standard methods for fixing and
staining adult midguts (see Note 3).
164
Jamie Adams et al.