Protocol for quantifying aberrant features of adult Drosophila midgut stem cells as tumor-prediction biomarkers

Summary

Here, we present a protocol for quantifying aberrant features of clustered intestinal stem cells (ISCs) in the adult Drosophila midgut, enabling tumor-prediction biomarker assessment. We describe steps for clustered ISC labeling, immunostaining, and cell number and size enumeration per midgut region during aging. We then detail procedures for determining mitosis rate and ploidy per clustered ISC. These measurements can be compared across different genotypes and experimental conditions and correlated with the corresponding late-in-life tumor incidence.

For complete details on the use of this protocol, refer to Neophytou et al.¹

Graphical abstract

Highlights

• •Multifaceted clustered stem cell analysis in the aging Drosophila midgut
• •Measurement of ISC-like cluster number and size per midgut
• •Measurement of mitosis rate and ploidy per clustered ISC-like cell
• •Steps for comparing across conditions and correlating with tumor incidence

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we present a protocol for quantifying aberrant features of clustered intestinal stem cells (ISCs) in the adult Drosophila midgut, enabling tumor-prediction biomarker assessment. We describe steps for clustered ISC labeling, immunostaining, and cell number and size enumeration per midgut region during aging. We then detail procedures for determining mitosis rate and ploidy per clustered ISC. These measurements can be compared across different genotypes and experimental conditions and correlated with the corresponding late-in-life tumor incidence.

Before you begin

Regenerative capacity is essential for maintaining tissue integrity during growth, aging, infection, and stress. The adult Drosophila midgut continuously renews itself via multipotent intestinal stem cells (ISCs), which divide asymmetrically or symmetrically.² ISCs express variable levels of Delta, a Notch pathway ligand. During asymmetric division, unequal distribution of Delta and other proteins in the two progeny cells results in one cell retaining the stem cell identity, while the other differentiates. In symmetric divisions, equal Delta distribution combined with Notch and other signaling determines whether both progeny will remain stem cells or differentiate.³ Most differentiating ISC progeny commit to the enteroblast (EB) fate, marked by the Su(H)GBE reporter expression, and subsequently become enterocytes. Alternatively, ISC progeny expressing Prospero eventually develop into enteroendocrine cells. Stomach (copper cell) region ISCs can give rise to additional cell types in the middle midgut.⁴ Interestingly, ISC progeny misdifferentiation, due to week Notch signaling, leads to aberrant ISC-like cell clustering, a type of progressive dysplasia.^1,5 High ISC-like clustering correlates with increased rate of DNA damage in Notch pathway gene loci leading to rare bona fide tumors in older flies^1,6

Innovation

Drosophila midgut studies often use progenitor cell counts and mitotic events per midgut as pathology readouts.⁷ While informative, these measurements provide limited insight into age-associated dysplasia and progenitor cell abnormalities. Here, we provide a nuanced progenitor cell analysis showing how to quantify various markers of aberrant ISC-like clusters as potential early-onset biomarkers that forecast late-life tumor incidence. We provide standardized criteria to identify and quantify ISC-like cluster number and cell composition per midgut region, as well as mitosis rate and endoreplication (ploidy) per clustered cell during aging and across different conditions. Moreover, correlation analyses may link early-life ISC-like clustering markers to tumor incidence.

Institutional permissions

Institutional approval for D. melanogaster research and biosafety regulations for handling genetically modified organisms is not necessary according to international guidelines. For standard wild-type and benign transgenic lines, research typically falls under Biosafety Level 1 (BSL-1) that requires basic containment, such as closed doors, screened windows, and mandatory freezing of fly morgues and cultures before disposal to prevent environmental escape or infestation.

Maintenance of fly stocks

• 1.
  Obtain the following Drosophila lines^1,6,8 (details in Table 1):

  • a.
    w; esg-Gal4 tub-Gal80^ts UAS-GFP Su(H)GBE-Gal80
  • b.
    w; UAS-GFP/CyO; Delta-Gal4/TM6C
  • c.
    ry⁵⁰⁶ Dl-lacZ⁰⁵¹⁵¹/TM3
  • d.
    w; Su(H)GBE-Gal4 UAS-GFP tub-Gal80^ts/TM6B
  • e.
    w; Su(H)GBE-GFP/TM6B
  • f.
    Oregon-R
  • g.
    Notch^55e11/FM7
  • h.
    Su(H)^del47/CyO
  • i.
    w¹¹¹⁸
• 2.Maintain fly stocks at 18°C with a 12:12h light:dark cycle and humidity at 60–70% to facilitate reproducibility.
• 3.
  Use fresh Drosophila fly food (stored at 4°C for up to 7 days). A standard recipe is the following (see Table 2):

  • a.
    Mix 10 g agar, 60 g cornmeal, 30 g inactivated dried yeast, and 50 g sucrose in 1 L of deionized water and heat to boiling point then remove from heating.
  • b.
    After cooling down to <80°C, add 5.8 mL of 20% Tegosept in 100% ethanol, and 3.8 mL 99% propionic acid, while stirring.
  • c.
    Pour fresh food into fly vials and let solidify and air dry for approximately 4 h at 20–30°C.

Table 1.

Useful Drosophila lines for ISC-cluster feature analysis

Drosophila line	Use	Source
w; esg-Gal4 tub-Gal80tsUAS-GFP Su(H)GBE-Gal80	ISCts-GFP, a temperature sensitive ISC marker and UAS transgene expression driver fully induced at 29°C	Used by Neophytou et al. iScience, 20251
w; UAS-GFP/CyO; Delta-Gal4/TM6C	Dl-GFP, ISC marker and UAS transgene expression driver	Used by Neophytou et al. iScience, 20251
ry506Dl-lacZ05151/TM3	Dl-lacZ, ISC marker	Used by Neophytou et al. iScience, 20251
w; Su(H)GBE-Gal4 UAS-GFP tub-Gal80ts/TM6B	EBts-GFP, a temperature sensitive EB marker and UAS transgene expression driver fully induced at 29°C	Used by Neophytou et al. iScience, 20251
w; Su(H)GBE-GFP/TM6B	EB-GFP, EB marker	Used by Neophytou et al. iScience, 20251
Oregon-R	OreR, wild type strain	Bloomington Drosophila Stock Center (2376)
Notch55e11/FM7	Create hemizygous females for spontaneous tumor analysis	Bloomington Drosophila Stock Center (#28813)
Su(H)del47/CyO or Su(H)2/CyO	Create hemizygous females for spontaneous tumor analysis	Bloomington Drosophila Stock Center (#30477)
w1118	Background for isogenization of UAS transgenes marked by mini-white gene	Bloomington Drosophila Stock Center (#3605)

Table 2.

Standard yeast-sucrose-cornmeal fly food ingredients

Fly food ingredient	Final concentration	Note
Yeast	30g/L (20-100g/L)	Bring to boiling point upon rigorous stirring
Cornmeal	60g/L	Bring to boiling point upon rigorous stirring
Sucrose	50g/L	Bring to boiling point upon rigorous stirring
Agar	10g/L	Bring to boiling point upon rigorous stirring
Tegocept	1.16mg/L	Add after cooling down to <80°C
Propionic acid	3.8ml/L	Add after cooling down to <80°C

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rabbit anti-phospho Histone H3 (Ser10) (1:200-4000)	Millipore	06-570; RRID: AB_310177
Donkey anti-rabbit Alexa FluorTM 488	Invitrogen	A21206; RRID:AB_2535792
Goat anti-chicken Alexa FluorTM 488	Invitrogen	A32931; RRID:AB_2762843
Donkey anti-mouse Alexa FluorTM 555	Invitrogen	A31570; RRID:AB_2536180
Donkey anti-rabbit Alexa FluorTM 555	Invitrogen	A31572; RRID:AB_162543
Chicken anti-GFP (1:2000)	Invitrogen	A10262; RRID: AB_2534023
Mouse anti-Delta (1:100)	DSHB	C594.9B; RRID: AB_528194
Mouse anti-Prospero (1:100)	DSHB	MR1A; RRID: AB_52844
Chemicals, petides, and recombinant proteins
DAPI	Sigma	D9542-10mg
Vectashield Mounting Medium	Vector Laboratories	H-1000
Hydroxyurea	Sigma Aldrich	H8627
Gemcitabine	Sigma Aldrich	G6423
Rapamycin	Sigma Aldrich	J62473.X3
Floxuridine	Sigma Aldrich	F0503
Bovine Serum Albumin	Sigma	A7888
Methanol	Honeywell	34860
Drosophila Agar Type II	Apex	66–103
Cornmeal	Sigma Aldrich	C6304
Sucrose	Sigma Aldrich	S7903
Tegosept	Apex	20–258
Propionic acid	Scharlau	AC18941000
Triton X-100	Sigma	T8787
Formaldehyde (4%)	Sigma	41116124
Other
Fly Incubators	Various suppliers	e.g., Phytotron or PHCbi
CO2 anesthesia system	Genesee Scientific	Bubbler kits, tubing, flypads, blowguns
Confocal microscope	e.g., Leica	e.g., TCS SP8 or STELLARIS
Fluorescent microscope	e.g., Zeiss	e.g., Axioscope A.1
Dissection stereoscope	e.g., Zeiss	e.g., Stemi
9-well plate	Corning	PYREX (No. Catalog No. 13-748B)
Tweezers	Fine Science Tools	Dumont #5 (No.11254-20)
ImageJ/Fiji	NIH	https://image.nih.gov/ij/
Graphpad Prism 9	GraphPad Software, Inc.	https://www.graphpad.com
Experimental models: Organisms/strains
Drosophila melanogaster: w1118	Bloomington stock center	6326; RRID:BDSC_6326
Drosophila melanogaster: Oregon-R	Bloomington stock center	2376; RRID:BDSC_2376
Drosophila melanogaster: Notch55e11/FM7	Bloomington stock center	28813; RRID:BDSC_28813
Drosophila melanogaster: Su(H)2/CyO	Bloomington stock center	30477; RRID:BDSC_30477
Drosophila melanogaster: w; esg-Gal4 tub-Gal80ts UAS-GFP Su(H)GBE-Gal80	Neophytou et al. iScience, 20251	NA
Drosophila melanogaster: w; UAS-GFP/CyO; Delta-Gal4/TM6C	Neophytou et al. iScience, 20251	NA
Drosophila melanogaster: ry506 Dl-lacZ05151/TM3	Neophytou et al. iScience, 20251	NA
Drosophila melanogaster: w; Su(H)GBE-Gal4 UAS-GFP tub-Gal80ts/TM6B	Neophytou et al. iScience, 20251	NA
Drosophila melanogaster: w; Su(H)GBE-GFP/TM6B	Neophytou et al. iScience, 20251	NA

Step-by-step method details

Clustered ISC-like cell labeling during aging

Timing: 1–3 months depending on the time points acquired

Use the following steps to produce genetically manipulated and environmentally treated flies with GFP-marked ISCs and ISC-like cells.

• 1.Cross virgin females of ISC^ts-GFP or an alternative ISC labelling genotype to OreR males to produce offspring bearing one copy of each transgene and no balancer chromosomes.

Note: Use 10 virgin females and 10 males per fly vial to be able to collect 20 male and 20 female progenies within 3 days of fly hatching.

Note: To do both ISC-like clustering and tumor incidence analysis cross ISC^ts-GFP to OreR or to Notch^55e11/FM7 or Su(H)^del47/CyO. Use 20–40 virgin females and 20–40 males per fly bottle to be able to collect 80 male and 80 female progenies without balancer chromosomes within 3 days of fly hatching. Depending on the useful portion of progeny, 1–3 bottles may be needed per experimental cohort.

Note: If flies of different UAS-RNAi lines need to be assessed, cross ISC^ts-GFP females to w¹¹¹⁸ control males and to w¹¹¹⁸;UAS-RNAi transgenic males of interest. The latter need to be isogenized by backcrossing to the w¹¹¹⁸ strain for ≥6 generations.

• 2.Incubate the progeny to adulthood for 10 days at 25°C. If UAS transgenes are included and need to be repressed via Gal80^ts during offspring development incubate the progeny to adulthood for 20 days at 18°C.
• 3.
  Collect and store at 18°C adult offspring for each experimental cohort every day for up to 3 days. From each cohort reserve 15 males and/or females for ISC-clustering analysis (Steps 22–39) and the rest for tumor incidence (Steps 40–45).

  • a.
    For ISC-clustering analysis collect 15 females and 15 males per condition in a fly vial.
  • b.
    To correlate ISC-clustering measurements with tumor incidence within replicate cohorts, collect ≥10 replicate cohorts of 200 males (or 50 females hemizygous Notch^55e11/+ or Su(H)^del47/+ females).
  • c.
    To compare ISC-clustering measurements and tumor incidence between genetically or conditionally different cohorts prepare 1 replicate cohort per condition.

Note: Spontaneous tumors develop in males that are naturally hemizygous for Notch and in hemizygous Notch^55e11/+ or Su(H)^del47/+ females.

CRITICAL: During fly collection minimize the time of CO₂ anesthesia of young offspring to ≤20 s to avoid stress.

• 4.Age females and males together for the first 4 days to ensure mating that is crucial for maximum ISC activity in females, but separate sexes thereafter.
• 5.Transfer flies to fresh standard or special diet food every 2 days at 25°C (or at 29°C to induce UAS transgenes that are repressed via Gal80^ts).

Note: At 25°C newly hatched flies are considered young for up to 7 days, middle aged between 14 and 21 days, and old beyond 30 days. At 29°C the corresponding age groups are 0–4, 10–15 and >20 days.

• 6.
  To decrease ISC-like clustering reduce yeast content to ≤2% or add drugs, such as the cell growth inhibiting, Rapamycin, or the DNA damage inducing, Floxuridine, in standard 3% yeast-sucrose-cornmeal food.¹ To increase ISC-like clustering increase yeast concentration in fly food to 10% or add DNA replication stress inducing drugs, such as Hydroxyurea or Gemcitabine, to standard food.¹ Take the following steps to add Hydroxyurea, Gemcitabine, Rapamycin or Floxuridine into standard fly food:

  • a.
    Dissolve 50 mg of Hydroxyurea in 1 mL of sterile distilled water to create a 50× stock solution (657 mM).
  • b.
    Dissolve 7.9 mg of Gemcitabine in 1 mL of distilled water to create a 100× stock solution (30 mM).
  • c.
    Dissolve 18.28 mg of Rapamycin in 1 mL of absolute ethanol to create a 100× stock solution (20 mM).
  • d.
    Dissolve 4.92 mg of Floxuridine in 1mL of distilled water to create a 100× stock solution (20 mM).
  • e.
    Warm standard cornmeal medium (Table 2) using microwave heating until completely liquefied for 2 to 3 min at 800W.
  • f.
    Dispense 2 mL of liquefied medium into each experimental vial. Then add the appropriate volume (20–40 μL) of stock solution containing a compound of interest in each vial, mix with a spatula and let solidify for about 2 hours.

Note: Feeding flies with virulent bacteria, such as Pseudomonas aeruginosa or with toxic agents (e.g. DSS, Bleomycin, pyocyanin, H₂O₂ or paraquat) can induce ISCs by damaging enterocytes or the ISC niche, per published methods.^9,10,11,12 Different treatments may affect ISC-like clustering with different kinetics, magnitude, or mechanism. The choice of treatment should depend on the biological impact of interest, and a pilot time-course and dose adjustment experiment might be needed (see Table 3).

Table 3.

Common treatments influencing ISC-like clustering

Treatment	Final concentration	Exposure window	ISC-like clustering
High Yeast	10% vs 2% in fly food	Throughout adult life	Induced (more nutrients)
Hydroxyurea	1 mg/mL in fly food	2 days	Induced (DNA replication stress)
Gemcitabine	30 μM in fly food	2 days	Induced (DNA replication stress)
Rapamycin	200 μM in fly food	Throughout adult life	Reduced (TOR inhibitor)
Floxuridine	200 μM in fly food	5–10 days	Reduced (DNA synthesis inhibition)
Pseudomonas aeruginosa	OD600nm ≤0.3 in 4% sucrose	1 to 2 days	Induced (primarily via enterocyte damage)
Bleomycin	≤2.5 μg/mL in 4% sucrose	1 to 2 days	Induced (via enterocyte damage)
Pyocyanin	50 mg/mL in 4% sucrose	1–5 days	Induced (primarily an ISC stimulus)
H2O2	≤1% in 4% sucrose	1–5 days	Induced (primarily an ISC stimulus)
Paraquat	≤1mM in 4% sucrose	1 to 2 days	Induced (enterocyte damage and ISC stimulus)
DSS	≤3% in 4% sucrose	1 to 2 days	Induced (ISC niche damage)

Midgut immunostaining

Timing: 2 days, including overnight incubation

Use the following steps to dissect and stain the Drosophila midgut so that GFP positive, mitotic and Prospero positive cells and their nuclei are fluorescently marked.

• 7.Prepare dissection solution: 1× PBS (130 mM NaCl, 70 mM Na₂HPO₄, 30 mM NaH₂PO₄, pH 7.4)
• 8.
  Set up dissection station:

  • a.
    Add 10 separate drops of 1× PBS, 50μL per drop, onto the Sylgard dissection plate (Petri dish filled with silicone elastomer). That is, one drop per fly to be dissected.
  • b.
    Place the Sylgard dissection plate and a multi-well plate on ice.
  • c.
    Prepare two sets of fine forceps and a dissection microscope.
• 9.Anesthetize flies briefly with CO₂ and transfer them on ice.

Transfer anesthetized flies one at a time under a dissecting scope next to a 50 μL drop of 1× PBS. Using a pair of forceps remove the fly head and gently detach the thorax from the abdomen. Then gently pull the anterior midgut out of the thorax and detach the posterior end of the abdomen to expose the hindgut. Gently pull the entire hindgut and midgut out of the fly body and inside the drop of PBS. Once inside the drop, remove the hindgut, crop, Malpighian tubules and ovaries.

Note: For more details on midgut dissection see published methods.^13,14

CRITICAL: Complete dissections within 30 min per 10–15 midgut group and keep dissected midguts on ice to preserve epithelial integrity.

• 10.Transfer all dissected midguts of the group in a well of a multi-well glass plate containing 300 μL of 4% formaldehyde in 1× PBS for 30 min at 20–30°C.
• 11.Rinse tissues 3 times quickly with 1× PBS (500 μL per wash).
• 12.
  When using anti-Delta antibody, continue fixation, as follows:

  • a.
    Add 1 mL of methanol (pre-cooled at −20°C) dropwise to tissues, that is, a 2:1 methanol:PBS ratio.
  • b.
    Replace with1 mL of 100% methanol and incubate for 5 minutes at room temperature.
  • c.
    Rehydrate by adding 1× PBS dropwise in a 1:2 ratio (methanol:PBS) for 20 s.
  • d.
    Rinse twice with 1× PBS for 20 seconds.
• 13.Incubate tissues in blocking solution (PBT: 1× PBS, 0.2% Triton X-100, 0.5% BSA) for at least 20 min at room temperature under gentle rocking.

Warning note: Methanol is hazardous and toxic. All procedures involving methanol must be performed in a certified chemical fume hood using a lab coat, safety goggles, and compatible gloves. Avoid inhalation, skin contact, and ignition sources.

• 14.
  Prepare primary antibody solution in PBT:

  • a.
    Rabbit anti-pH3 (1:200–4000) to detect cells undergoing mitosis.
  • b.
    Chicken anti-GFP (1:2000) to enhance GFP detection in GFP-expressing flies.
  • c.
    Mouse anti-Delta (1:100) to detect ISCs and ISC-like cells.
  • d.
    Mouse anti-Prospero (1:100) to detect EEs.

Note: anti-Delta staining is an alternative to anti-GFP to mark ISCs and ISC-like cells, when using flies lacking the ISC^ts-GFP transgenes.

• 15.Incubate tissues in primary antibody solution overnight at 4°C under gentle rocking.

Note: Use 300 μL antibody solution per 10-15 midguts. Ensure tissues are fully submerged in the wells of the multi-well glass plate.

• 16.Wash tissues 3 times for 10 minutes each with PT solution (1× PBS, 0.2% Triton X-100) at room temperature with gentle rocking.
• 17.
  Prepare secondary antibody solution in PBT with DAPI:

  • a.
    Alexa Fluor 488 or 555 conjugated secondary antibodies (1:1000).
  • b.
    DAPI (1:3000 from 10 mg/mL stock).
• 18.Incubate tissues in secondary antibody solution for 1 to 2 h at room temperature with gentle rocking, protected from light.
• 19.Wash tissues 3 times for 10 minutes each with PT solution at room temperature with gentle rocking.
• 20.
  Mount tissues on glass slides:

  • a.
    Place 15–20 μL Vectashield mounting medium on slide.
  • b.
    Carefully transfer 5–7 midguts per slide.
  • c.
    Use forceps to disentangle and roughly arrange midguts parallel to each other.
  • d.
    Cover with 22 × 22 mm coverslip, avoiding trapping bubbles underneath.
  • e.
    Seal edges with nail polish if storing long-term.
• 21.Store slides at 4°C protected from light until imaging for up to 1 week or at −20°C for long term storage.

Imaging and ISC-like cluster analysis

Timing: ∼2 days for imaging and quantification

Use the following steps to image Drosophila midgut ISCs and ISC-like cells and quantify their features.

Quantification of ISC-like cluster number and size per midgut region

• 22.Define ISC-like clusters manually using a fluorescent microscope by locating groups of 3–20 adjacent cells expressing ISC markers (ISC^ts-GFP or Dl expression) (Figure 1A).

Note: Clusters are distinguished based on the rule of 0-1 cell border sharing. Moreover, clustered cells and their DAPI-marked nuclei are heterogeneous in size.

• 23.Count the number of clusters per midgut or midgut region of interest (e.g., A1, P1, P4) while stratifying each cluster according to its number of cells (3, 4, 5, 6, 7, or ≥8).
• 24.Plot the frequencies of clusters per midgut or midgut region. Stack frequencies according to the number of cells per cluster (Figure 1B). If stratified cluster frequency needs to be statistically compared between conditions, apply the Chi-square test using a 6 × 2 contingency table (6 strata × 2 observed raw values for each).

Figure 1.

Assessment of ISC-like cluster number and size

(A) Outlined ISC-like clusters in the posterior (P4) midgut of ISC^ts-GFP females. Adapted from Neophytou et al.¹ Scale bar, 37.5 μm.

(B) Plot of frequencies of ISC-like clusters per midgut stacked per cluster size of 4- and 30-day old females and males. Error bars represent standard deviation.

Quantification of the average mitosis rate per clustered ISC-like cell

• 25.Define ISC-like clusters per whole midgut or midgut region of interest.
• 26.Count the number of pH3 positive cells per cluster, while stratifying each cluster according to its number of cells (1–20) (Figure 2)
• 27.Calculate the mitotic index of each cluster, as follows: 100× pH3+ cells in a cluster/number of cells in that cluster.
• 28.Plot the series of average mitotic indices per cluster size (Figure 2A). To statistically compare the average mitosis indices between conditions, apply the Chi-square test using e.g., a 14 × 2 contingency table for 14 strata × 2 conditions.

Figure 2.

Quantification of clustered ISC-like cell mitosis rate

(A) Plot of the mitotic index of single and double ISCs and clustered ISC-like cells stratified according to the number of cells per cluster in the P4 region of 30-day old females. Adapted from Neophytou et al.¹ Error bars and asterisks indicate standard deviation and p-value <0.05, respectively.

(B) Image of single and double ISCs and clustered ISC-like cells (green), some of which are positively marked for pH3 (red), as indicated with the yellow arrowheads.

Quantification of ISC-like cell ploidy

• 29.
  Set up a confocal microscope with appropriate laser wavelength and filters:

  • a.
    405 nm laser for DAPI.
  • b.
    488 nm laser for GFP or anti-GFP Alexa Fluor 488.
  • c.
    555 nm laser for Alexa Fluor 555 (binding any additional primary antibody).
• 30.Use 40 × oil immersion objective with 1 × zoom for optimal resolution.
• 31.
  Acquire Z-stacks of midgut regions spanning the entire depth of the nuclei of the upper monolayer of cells (epithelial cells closest to the lenses):

  • a.
    10-15 optical sections spanning the entire epithelial thickness.
  • b.
    Z-step size: 1 μm.
  • c.
    Image format: 1024 × 1024 pixels.
  • d.
    Frame size: 375 × 375 μm.
• 32.
  Specify midgut regions, for example:

  • a.
    Posterior A1 (R1b) – coldspot region for ISC-like clusters.
  • b.
    P1 (R4a) – hotspot region for ISC-like clusters.
  • c.
    Posterior P4 (R5b) – hotspot region for ISC-like clusters.

CRITICAL: Ensure images are acquired using identical microscope and scanning settings for all samples to enable quantitative comparisons. Use identical laser power, gain, and offset settings that lie below signal saturation for all images to be compared.

• 33.Generate projections from 10–15 serial optical sections covering the full depth of nuclei monolayer closest to the lenses.
• 34.Export images in TIFF format as maximum intensity projections for DAPI-marked nucleus area measurements, a proxy for DNA content, or as sum projection for direct DAPI-stained DNA quantification.

Note: Measure (a) DAPI-marked nuclear area using maximum intensity projection images and/or (b) integrated DAPI intensity per nucleus using sum projection images.

• 35.Open DAPI-stained midgut images in ImageJ/FIJI software.
• 36.
  Mark all clustered ISC-like cells (using the GFP channel, as in Figure 1A) and 12 small single ISCs as control diploid cells per image. Then use the DAPI channel of the same image (as in Figure 3A) and the oval selection tool on the menu bar (screenshot provided) to manually outline the maximum cross-sectional area of each nucleus.

  

• 37.
  Measure and export in xls the DAPI nuclear area (or DAPI intensity) of all clustered ISC-like cell nuclei, and of the 12 small single ISCs per image using the “Analyze” tool on the menu bar (screenshot provided)

  

• 38.Calculate the ploidy (genome copy number) per midgut image, as follows: 2 × DAPI nuclear area (or signal intensity) per clustered ISC-like cell nucleus/mean DAPI nuclear area (or DAPI intensity) of the 12 single ISC nuclei.
• 39.Plot the average ploidy of all ISC-like cells or the average ploidy stratified into sequential groups, e.g., 0–3, >3–6, >6–10, >10–14, >14–18, >18–22 etc. (Figure 3B). To statistically compare the average ploidy between conditions, apply the Chi-square test using e.g., a 6 x 2 contingency table for 6 strata x 2 conditions. Also plot the average ploidy of the single ISCs, which are defined as diploid (2C) on average.

Figure 3.

DAPI-based ploidy quantification of ISC-like cells

(A) Clustered ISC-like cells (marked based on the GFP channel as in Figure 1A) exhibit small and larger DAPI-stained nuclei (white), indicating increased ploidy.

(B) Plot of the average ploidy of clustered ISC-like cells stratified into six sequential ploidy groups, 0–3, >3–6, >6–10, >10–14, >14–18, >18–22. Adapted from Neophytou et al.¹

Spontaneous age-related tumor incidence measurement

Timing: ∼2 days for imaging and quantification of flies at an old age time point, when survival is still ≥90%. For example, at 42 days, if OreR flies are incubated at 25^oC

Use the following steps to measure ISC and Prospero tumor incidence.

• 40.Prepare ≥180 males naturally hemizygous for Notch or ≥35 Notch^55e11/+ or Su(H)^del47/+ females per experimental condition, as described in Step 1.

Note: Use flies from the same cohort and sex group as those for ISC-like cluster analysis to later correlate with ISC-like cluster features (Steps 46–54).

• 41.Age flies at identical densities, diet, and other environmental parameters across all conditions to be compared by transferring ∼60 flies per bottle or ∼15 flies per vial at the same time using the same media and incubators.

Note: Transfer flies to fresh food every 2 days for the first half of their life and every day thereafter to minimize microbial contamination. Avoid CO₂ anesthesia after initial cohort setup to prevent unnecessary stress.

• 42.Perform immunostaining as described in Steps 7–21.
• 43.Define ISC and EE tumors manually using a fluorescent microscope by locating groups of ≥80 adjacent diploid-looking cells expressing ISC markers (ISC^ts-GFP, Dl-GFP expression or anti-Dl staining) and/or the EE marker Prospero (Figure 4A).

Note: Unlike the ISC-like cluster cells (Figure 1A), ISC and EE tumor cells are diploid and homogeneous in the size. Moreover, tumors are relatively rare (Figure 4B), much bigger and appear late in life.

• 44.Count the number of ISC and EE tumors per midgut or midgut region of interest (e.g., A1, P1, and P4).
• 45.Plot the % of ISC and EE tumors per midgut or midgut region (Figure 4B). To statistically compare tumor incidence between conditions, apply the Chi-square test using a e.g., 2 × 2 contingency table for 2 possible outcomes × 2 conditions tested.

Figure 4.

Tumor incidence assessment and correlation with ISC-like cluster number

(A) Indicative ISC tumor surrounded by ISCs and ISC-like clusters marked by Dl-GFP (Dl-GFP crossed to OreR).

(B) Tumor incidence (%) in different genetic backgrounds (Dl-GFP crossed to w¹¹¹⁸, or OreR or the Drosophila Genetic Reference Panel lines 28194 and 28217). n=691–754 midguts tested per condition.

(C) Correlation of the total Dl-GFP clusters per midgut at 4 days with tumor incidence (%) at 42 days within n=15 replicate cohorts. Adapted from Neophytou et al.¹ p-value <0.0001.

Comparative ISC-like cluster and tumor measurements

Timing: ∼1 day (data organization, statistical analysis, and visualization)

Use the following steps to correlate ISC-like cluster measurements with tumor incidence within replicate cohorts, as well as, to compare ISC-clustering measurements and tumor incidence between conditionally different cohorts.

Data assembly

• 46.
  Prepare ≥ 10 independent replicate cohorts, as described in Step 3, to obtain early life ISC-like clustering measurements for one or more of the following features per midgut (or midgut region) for each cohort:

  • a.
    Total cluster number, i.e., sum of all clusters irrespective of size.
  • b.
    Total clustered cells, i.e., sum of each cluster × number of cells in it.
  • c.
    Average clustered cell mitosis rate, i.e., average mitosis of all clustered cells.
  • d.
    Average clustered cell ploidy i.e., average ploidy of all clustered cells.

Note: To correlate ISC-like cluster measurements with tumor incidence within replicate cohorts, use ≥10 replicate cohorts of 200 males (or 50 females hemizygous Notch^55e11/+ or Su(H)^del47/+ females). To compare ISC-clustering measurements and tumor incidence between conditionally different cohorts use 1 replicate cohort per condition. Although ISC-clustering measurements at any time point can be assessed, early-life time points of 4–7-days old flies are of predictive value.

• 47.Spare flies of the independent replicate cohorts described in the previous step to obtain the corresponding late in life ISC and/or EE tumor incidence, i.e., the % of tumors per midgut or midgut region described in Steps 40–45.

Correlation of ISC feature measurements with tumor incidence within replicate cohorts using GraphPad

• 48.Select one of the 4 features at a time, either total cluster number, total clustered cells, average clustered cell mitosis rate, or average clustered cell ploidy.
• 49.Match and corelate any ISC-like feature measurement of interest with the corresponding tumor incidence measurement for every replicate cohort per sex using Pearson correlation for normally distributed data or Spearman correlation for non-parametric data.
• 50.Plot the linear relationship between the two continuous variables (one ISC feature measurement per replicate cohort with the matching tumor incidence) (Figure 4C).
• 51.Report the correlation coefficients (r) and associated p-values, that measure the strength and direction of a linear relationship between the two continuous variables (ISC feature measurement and tumor incidence).
• 52.Report the coefficients of determination, R², that represent the proportion of the variance in the dependent variable (tumor incidence) that is predictable from the independent variable (ISC feature measurement).

Comparing ISC-cluster measurements between conditionally different cohorts anticipating differences that reflect on tumor incidence

• 53.Genetic, dietary or exposure conditions directly modulating an ISC-like feature may be assessed to causally link that feature to tumor incidence. To do so, measure the impact of modulating an ISC feature of interest using transgenes, diets or drugs known to affect that feature per Steps 22–38, and the corresponding tumor incidence per Steps 40–44.
• 54.Compare the measurements between control and test cohort for the ISC-like feature of interest, per Steps 24, 28 or 39, and tumor incidence, per Step 45.

Expected outcomes

ISC-like cluster formation

Young flies (4–7 days): Expect 2–5 clusters of 3–6 cells per midgut, predominantly in P1 and P4 regions. A1 region should show minimal clustering. Old flies (≥30 days): Expect 6–15 clusters per midgut, with larger clusters (7–16 or more cells) becoming more frequent, especially in posterior regions. Sex differences: Females should show approximately 3 times more clusters than males at late time points.

ISC-like cell ploidy

Young flies (4–7 days): ISC-like cells typically exhibit low to moderate DAPI signal increase in ploidy. Expect predominantly diploid or mildly polyploid nuclei and a minimal variation between clustered and single ISC-like cells. Aged flies (≥30 days): ISC-like clustered cells show a significant increase in normalized DAPI integrated intensity, indicative of increased ploidy, particularly in posterior midgut regions (e.g., P4). Cluster association: Ploidy is higher in clustered ISC-like cells compared to single ISCs, with larger clusters displaying greater variability in nuclear size and DNA content. Predictive relationship: Increased endoreplication in early-life ISC-like clusters might positively correlate with cluster expansion and in turn with tumor incidence at later time points.

While clustered cells are generally heterogeneous in size, ISC and EE tumor cells are not.

Drug-induced ISC-like clustering

Gemcitabine (30 μM, 2 days): Tentatively increases ISC-like cell clustering in young females and males. Hydroxyurea (1 mg/mL, 2 days): Tentatively increases ISC-like cell clustering in both sexes. 10% vs 2% yeast in yeast-sucrose-cornmeal diet throughout adult life: Increases ISC-like cell clustering in females.

Drug-suppressed ISC-like clustering

Rapamycin (200 μM): Reduces ISC-like cell clustering more clearly when administered early in life (days 0–21). Floxuridine (200 μM): Reduces clustering more clearly when administered early in life (days 0-5).

Comparisson of ISC features early and late in life

High clustering at days 4–7 is indicative of elevated ISC-like clustering at later timepoints (days 20–30). Intervention window: ISC-like clustering reduction interventions before midlife (e.g., day 21 at 25^oC) are most effective at reducing late-life clustering.

Correlation of ISC features with tumor incidence

Increased tumor incidence late in life is typically associated with increased ISC cluster number and size early in life, reflecting compromised ISC homeostasis. Conditions that suppress ISC clustering are expected to delay age-associated tumor formation.

Limitations

Marker specificity: ISC^ts-GFP marks both true ISCs and ISC-like cells. Distinguishing between them requires analysis beyond clustering and ploidy assessment, such as, co-expression of the ISC marker, Delta (some ISC^ts-GFP cells do not express the ISC marker Delta even in young flies), the EB marker, Su(H)GBE-GFP (many clustered Dl+ cells have been shown to also express the EB marker, Su(H)GBE-GFP) or the EE marker, Prospero (very few ISC^ts-GFP cells have been shown to express this enteroendocrine marker).

Temperature sensitivity: the Gal80^ts system requires precise temperature control. Temperature fluctuations and Drosohila line variations in transgene expression levels affect its efficacy.

Regional variation: Cluster frequency varies significantly by midgut region. Compare matched regions only between experimental groups.

Genetic background effects: ISC-like clustering is a quantitative trait influenced by the genetic background. Use isogenic controls when comparing different fly genotypes.

Fixation artifacts: Methanol fixation (required only for anti-Dl antibody staining in this protocol) may alter tissue morphology slightly. Include non-methanol controls when assessing cell architecture.

Imaging depth: Confocal penetration may be limited to ∼50 μm. In thick (non-flattened) midgut mounting deep epithelial regions may be under-represented in the analysis.

ISC feature - tumor incidence correlations within replicate cohorts: These do not demonstrate causal link, plus they are laborious requiring ≥ 10 independent replicate cohorts to test the correlation of an ISC feature with tumor incidence. Genetic, dietary or drug exposure manipulations that specifically target an ISC feature can be used to establish causality. For example, use the ISC^ts-Gal4 and EB^ts-Gal4 drivers to reduce progenitor cell mitosis via cycE RNAi in ISCs, endoreplication via cycE RNAi in EBs, and ISC progeny differentiation via Notch RNAi in EBs comparing tumor incidence in 1 test/RNAi versus 1 control cohort.

Troubleshooting

Problem 1

• •No GFP signal visible (related to steps 1–5 and 29–33).
• •Possible cause: (a) Gal80^ts not properly inactivated, (b) incorrect genotype, (c) imaging settings too low.

Potential solution

• •Raise flies at 29°C for ≥24h before dissection, if tub-Gal80^ts is used, and/or use anti-GFP staining.
• •Select against balancer marker bearing flies carefully or obtain a new stock.
• •Increase laser power and PMT gain; check with positive control.

Problem 2

• •Very few or no clusters detected (related to steps 1–6 and 22 to 23).
• •Possible cause: (a) flies too young, (b) wrong midgut region imaged, (c) low mitotic genetic background.

Potential solution

• •Use flies ≥4 days old as clustering increases with age.
• •Study the posterior P1 and P4 hotspot regions, rather than the cold-spot anterior region A1.
• •This is normal for some genetic backgrounds, e.g., w¹¹¹⁸. Use OreR for higher baseline.

Problem 3

• •High background fluorescence (related to steps 9–21).
• •Possible cause: (a) insufficient washing, (b) antibody concentration too high, (c) tissue degradation.

Potential solution

• •Increase wash times to 15 min each, use fresh PT solution.
• •Reduce antibody concentration (test serial antibody dilutions).
• •Work more quickly during dissection and keep tissues on ice for up to 30 min between dissection and fixation.

Problem 4

• •Poor tissue morphology (related to steps 9–21).
• •Possible cause: (a) over-fixation, (b) tissue damage during dissection, (c) methanol treatment too harsh (required for anti-Delta only).

Potential solution

• •Fix for 30 min or less.
• •Practice gentle handling without pulling or pinching the tissues with the forceps.
• •Ensure gradual transitions during methanol dehydration/rehydration.

Problem 5

• •Cannot distinguish ISC-like clusters from tumors (related to steps 22–24).
• •Possible cause: (a) not following rigorous cluster/tumor defining criteria, (b) tumor prone genotype or condition.

Potential solution

• •ISC-like clusters are frequent in young and very frequent old flies. They exhibit 3–20 cells of variable ploidy. When too many per midgut region as in old highly mitotic flies they may merge with each other as well as with tumors that are very rare in principle and present only in old flies. Tumors are composed of ≥80 cells, uniform in ploidy cells.
• •Oncogene expression and tumor suppressor loss of function drives ISCs tumors even in young flies that may saturate the epithelium within a week rendering meaningful tumor rather than cluster analysis.

Problem 6

• •High variability between biological replicates (related to steps 1–6)
• •Possible cause: (a) inconsistent fly fitness, (b) regional variation, (c) microbial contamination.

Potential solution

• •Collect the first progeny of the youngest parents, i.e., the progeny that become adults first during the first 3 days of adult progeny collection.
• •Always quantify the same midgut regions (e.g., A1, P1, P4) across samples.
• •Transfer parental crosses to fresh fly food every day and add broad spectrum antibiotics to the fly food for 4 days to clear parents and progeny from excessive bacterial load.

Problem 7

• •Cannot distinguish ISC-like clusters from one another (related to steps 22–24).
• •Possible cause: (a) clusters are too dense, (b) laser power or signal amplifier (PMT) gain too high.

Potential solution

• •Select an earlier time point or clean microbial contamination.
• •Group cells into smaller loosely attached clusters.
• •Reduce laser power or PMT so that the brightest sample does not exhibit saturated (max intensity) pixels.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Yiorgos Apidianakis (apidiana@ucy.ac.cy).

Technical contact

Further information on executing this protocol should be directed to and will be answered by the technical contact, Yiorgos Apidianakis (apidiana@ucy.ac.cy).

Materials availability

Drosophila strains mentioned in this study are available upon request from the lead contact.

Data and code availability

All data supporting the findings of this study are available from the lead contact upon request.

Acknowledgments

This work was supported by funding from the Republic of Cyprus, “Restart 2016–2020 Programmes,” through the Research and Innovation Foundation (LDDTA Project: EXCELLENCE/0421/0323).

Author contributions

S.T. wrote the paper. S.T. and Y.A. edited the paper. Y.A. prepared responses to editors and reviewers.

Declaration of interests

The authors declare no competing interests.