2.1 Cell Culture
T84 and LoVo cells were purchased from American Type Culture Collection (ATCC) (Middlesex, UK), Caco-2 cells were purchased from ŞAP Enstitüsü (Ankara, Turkey), and HCT-116 cells were purchased from DKFZ (Heidelberg, Germany). HEK293 cells were obtained as a kind gift from Dr. İhsan Gürsel (Izmir Biyotip ve Genom Merkezi, Izmir), and HeLa cells were a kind gift from Prof. Dr. Ayşe Elif Erson Bensan (METU, Ankara). T84 cells were cultured in DMEM-HAMS F12 (Biological Industries) medium supplemented with 10% fetal bovine serum (FBS; Biological Industries). Caco-2 cells were maintained in Eagle’s minimum essential medium (EMEM) supplemented with 20% FBS (Biological Industries) and 1 mM sodium pyruvate (Biological Industries). LoVo, HCT-116 and HEK293 cells were cultured in RPMI 1640 medium (Biological Industries) supplemented with 10% FBS (Biological Industries). HeLa cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10% FBS (Gibco). All culture media were supplemented with 2 mM L-glutamine (Biological Industries) and 100 U/mL penicillin − 100 µg/mL streptomycin (Biological Industries), and the cells were cultured in an incubator with 95% air and 5% CO2 at 37°C. The cells were routinely tested for mycoplasma contamination by PCR.18 Plasmocin (Invitrogen) was added to the culture medium at a concentration of 2.5 mg/ml to prevent any mycoplasma contamination.
2.2 Nutrient Depletion Protocol
T84, Caco-2, and HeLa cells were grown in glucose and glutamine-free DMEM (Biological Industries), while LoVo, HCT-116, and HEK293 cells were grown in glucose and glutamine-free RPMI. Both media were supplemented with 1% FBS (Biological Industries), 0.2 mM L-glutamine (Biological Industries), 0.1 g/L glucose, and 100 U/mL penicillin − 100 µg/mL streptomycin (Biological Industries). The cells were incubated for 48 h in nutrient-depleted medium unless otherwise stated. For replenishment studies, after a 48 h incubation of the cells in the nutrient depletion medium, the cells were washed with phosphate buffered saline (PBS, Biological Industries) and incubated in complete medium for 24 h.
2.3 Drug/Chemical Treatments
The drugs and chemicals used in the current study are shown in Table 1. For drug treatments of 24 h duration, the cells were first incubated in nutrient-depleted medium for 24 h followed by treatment in nutrient-depleted medium for an additional 24 h. The total duration of treatment or nutrient depletion did not exceed 48 h.
Table 1
Chemicals used in the study.
Chemical | Concentration & Treatment Duration | Vehicle | Brand & Catalog Number |
5-Fluorouracil (5-FU) | 50µM 48 h | DMSO | Sigma Aldrich 51-21-8 |
Doxorubicin hydrochloride | 10µg/mL 24 h and 48 h | DPBS | Sigma Aldrich 25316-40-9 |
Bafilomycin A1 | 100 nM 48 h | DMSO | InvivoGen 88899-55-2 |
3-Methyladenine (3-MA) | 5 mM 48 h | DPBS | Sigma Aldrich 5142-23-4 |
Cycloheximide (CHX) | 20µg/mL 24 h | DPBS | Sigma Aldrich 66-81-9 |
MG-132 | 10µM 24 h | DMSO | Tocris 1748 |
Sodium Arsenite | 200µM 1 h | DPBS | Sigma Aldrich S7400 |
2.4 RNA Isolation, cDNA Synthesis and qRT‒PCR
Total RNA was isolated using the NucleoSpin RNA Kit (Macherey Nagel) according to the manufacturer's recommendations and stored at -80°C until use. cDNA was synthesized from 1-2.5 µg RNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) with the use of random hexamers and was stored at -80°C until use. qRT‒PCR was performed in a Rotor GeneQ 6000 series (Qiagen) with Loading Block 72. In total, a 10 µL reaction mix was prepared containing 5 µl of 2X GoTaq qPCR Master Mix (Promega), 2 µM forward and reverse primers, and 2 µl of cDNA. Standard curves for each primer pair were generated. Threshold cycle (Ct) values were determined after 45 cycles using the relative standard curve method. The Pfaffl method was used to calculate the fold change in expression.19ACTB was used as the housekeeping gene. The sequences of the primers are shown in Table 2.
Table 2
Sequences of primers used in the study.
Target Gene | Forward and Reverse Primers | Annealing Temperature | Product Size |
ACTB NM_001101.5 | 5’TGTCCACCTTCCAGCAGATGT 5’AGCTCAGTAACAGTCCGCCTAGA | 50–60°C | 101 bp |
SQSTM1 NM_001142298.2 NM_001142299.2 NM_003900.5 | 5’ATGAGGACGGGGACTTGGTT 5’TTGCAGCCATCGCAGATCA | 57°C | 190 bp |
LAMP1 NM_005561.4 | 5’ATGTGTTAGTGGCACCCAGG 5’TGTTCACAGCGTGTCTCTCC | 56°C | 117 bp |
MCOLN1 NM_020533.3 | 5’CATGAGTCCCTGCGACAAGT 5’ACCACGGACATACGCATACC | 60°C | 310 bp |
RAB7A NM_004637.6 | 5’AGTGTTGCTGAAGGTTATCATCC 5’TTCCTGTCCTGCTGTGTCC | 56°C | 187 bp |
MIR143 Gene NC_000005.10 | 5’ TTGAATTCCCCTCTAACACCCCTTCTC 5’ TTAAGCTTAACTTCCCCAGCATCACAAG | 63 oC | 276 bp |
2.5 Isolation of miRNA and miRNA qRT‒PCR
mirRNAs were isolated from 48h nutrient-depleted or nutrient-rich Caco-2 cells using the mirVAna miRNA Isolation Kit (Thermo Fisher Scientific) in accordance with the protocol suggested in the kit and sequenced for small RNAs at Beijing Genome Institute (Hong Kong). The list of differentially expressed miRNAs is shown in Supplementary Table 1. To determine miR143-3p levels in T84 cells, a qRT‒PCR protocol was applied as described previously.20 In this method, reverse transcription uses the enzyme PolyA Polymerase to increase the length of mature miRNAs with a Poly A tail. The forward sequence contains sequences specific to the individual miRNAs.21 The reverse transcription (RT) primer used contains an adapter sequence conjugated to a primary oligodT sequence along with the miRNA specific sequence.21
The reaction mixture consisted of 100 ng cDNA, 1 µL of 10X E. coli poly(A) polymerase Reaction buffer, 5 U/mL Polymerase A (NEB), 0.1 mM ATP (NEB), 1 µM RT primer, 0.1 mM dNTP mix (Thermo Fisher Scientific) and 5 U/mL Revert-Aid First Strand Reverse Transcriptase (Thermo Fisher Scientific). The total volume was kept at 10 µL using nuclease-free water. qRT‒PCR was performed as described above. The hsa-let7a primer pair was used as an internal control since its levels were found not to change with nutrient depletion. The primer pairs used to amplify the microRNAs are shown in Table 3.
Table 3
Primer pairs used in the miRNA-specific qRT‒PCR
Target miRNA | RT Primer | Forward and Reverse Primers |
hsa-let7a | CAGGTCCAGTTTTTTTTTTTTTTCT | 5’GCAGTGAGGTAGTAGGTTGT 5’GGTCCAGTTTTTTTTTTTTTTTAACTATAC |
hsa-miR-143-3p | 5’GCAGTGAGATGAAGCACTG 5’TCCAGTTTTTTTTTTTTTTTCTCGA |
2.6 Plasmids and cloning
A Nanoluc luciferase reporter assay was carried out to confirm the post transcriptional targeting of Rab7a by miR-143. For this, the 3'UTR of RAB7A was cloned in the pGWIZ RPS30M SuperNanoLuc luciferase reporter vector (obtained as a kind gift from Dr. Khalid S Khabar, King Faisal University, Saudi Arabia). Normalization was carried out with the pGWIZ RPS30M Firefly vector. For overexpressionf of hsa-miR-143, the sequence was amplified from HEK293 cells using primers (Table 2) with Q5 High-Fidelity DNA Polymerase (NEB), purified with gel purification and digested with EcoRI and HindIII. The sequences were ligated to the digested pcDNA3.1/Neo vector (kind gift from Prof. Dr. Elif Erson Bensan, METU, Ankara) using T4 DNA Ligase (NEB) and T4 DNA Ligase Buffer (NEB) overnight at 16°C, transformed into DH5α competent E. coli and incubated at 37°C until colonies appeared on an ampicillin plate. The cloning was confirmed by Sanger sequencing. Transient transfection of plasmids was performed using the FuGENE HD (Promega) transfection reagent at a ratio of 2:1 (transfection reagent to plasmid DNA) in 48-well plates. A total of 62.5 ng of the SuperNanoLuc reporter vector, 562.5 ng of the Firefly vector and 625 ng of the miR-143-overexpressing pcDNA3.1 vector were used for transfection. The transfection was carried out for 24 h, after which the cells were collected. Cells were lysed as specified in the manual foe Nano-Glo Dual Luciferase Reporter Assay System (Promega), and Firefly and NanoLuc luciferase activities were determined using the SpectraMax iD3 Multimode Microplate Reader (Molecular Devices).
2.7 siRNA-mediated silencing of RAB7A
A pool of 4 siRNAs targeting RAB7A (Dharmacon) along with its scrambled siRNA control was used. HeLa cells in complete medium were seeded into 6-well plates at a confluency of 70–80% and left to attach overnight. For each well, a mixture of siRNA (10 nM) and transfection reagent (3 µl) was prepared in Opti-MEM reduced serum medium (Gibco) to a final volume of 100 µl and incubated at room temperature for 10 min. The cells were washed once with PBS (Biological Industries), and 1 mL of Opti-MEM reduced serum medium (Gibco) was added to each well, followed by the addition of the transfection mix in a dropwise manner. The cells were incubated for 24 h and then processed for western blotting.
2.8 Protein Isolation and Western Blot
2.8.1 Total Protein Isolation
Cell pellets were collected and washed with ice-cold PBS and centrifuged at 1000 x g for 5 minutes at 4°C. Total protein was isolated using the Mammalian Protein Extraction Reagent M-PER (Thermo Fisher Scientific) according to the manufacturer's instructions, supplemented with PhosSTOP Phosphatase Inhibitor (Roche) and complete Mini EDTA-free Protease Inhibitor Cocktail (Roche). Isolated protein samples were stored at -20°C until use. Proteins were quantified with a Coomassie Reagent using the Bradford assay.
2.8.2 Cytoplasmic and Nuclear Protein Isolation
Cell pellets were washed twice with 0.3 ml of hypotonic PBS and centrifuged at 500 x g for 5 min at 4°C. The lysed cells were combined with 75 µl of 10% NP40 (AppliChem) and incubated on ice for 15 minutes. The tubes were centrifuged at the maximum speed for 30 seconds at 4°C. The supernatant, which contained cytosolic proteins, was transferred to a fresh centrifuge tube. The remaining pellet was resuspended in 80 µl of nuclear extraction buffer. The mixture was vortexed for 30 seconds and incubated on ice for 15 minutes on an orbital shaker, and this cycle was repeated one more time. The mixture was centrifuged at 14000 x g for 10 minutes at 4°C, and the supernatant containing the nuclear fraction was collected in a fresh centrifuge tube.
2.8.3 Western blot
Western blotting was carried out according to well-established protocols using 20–30 µg of protein. The antibodies used in the study are shown in Table 4. Since the expression of a number of proteins was determined from the same blot, we cut the membrane into two or three pieces before incubation with the primary antibody. The full blots for these two/three pieces of the membrane are shown in Source Data files. Mild stripping (1.5% glycine, 0.1% SDS, 1% Tween-20) was performed at 60°C for 5 minutes, and reprobing was applied when necessary. Antibodies raised in different animals were selected when detecting proteins of comparable sizes. Densitometric analyses of the blots were carried out using Image Lab Software (Bio-Rad).
Table 4
Antibodies used in the current study.
Antibody | Size | Origin | Dilution | Brand and Catalog # |
Antibodies for western blot |
AMPKα (T172) | 62 kDa | Rabbit | 1:1000 | Cell Signaling Technologies, 2535S |
AMPKα1/2 | 62 kDa | Mouse | 1:1000 | Santa Cruz Biotechnology, sc-74461 |
p70S6K (49DS7) | 70–85 kDa | Rabbit | 1:1000 | Cell Signaling Technology, 2708P |
p70S6K (T389) | 70–85 kDa | Rabbit | 1:1000 | Cell Signaling Technology, 9234S |
4E-BP1 (Ser65) | 12–20 kDa | Rabbit | 1:1000 | Cell Signaling Technology, 9451S |
4E-BP1 (53H11) | 12–20 kDa | Rabbit | 1:1000 | Cell Signaling Technology, 9644T |
p62/SQSTM1 | 62k Da | Mouse | 1:1000 | Santa Cruz Biotechnology, sc-5286 |
LC3 A/B (D3U4C) | 14–16 kDa | Rabbit | 1:1000 | Cell Signaling Technology, 12741S |
LAMP1 (H4A3) | 110 kDa | Mouse | 1:1000 | Santa Cruz Biotechnology, sc-20011 |
Rab7a | 23 kDa | Rat | 1:1000 | BioLegend, 850401 |
TF-EB | 65–70 kDa | Mouse | 1:1000 | BioLegend, 852001 |
P-RPS6 (Ser235/236) | 32 kDa | Rabbit | 1:1000 | Cell Signaling Technology, 4858T |
GAPDH (FL-335) | 37 kDa | Rabbit | 1:4000 | Santa Cruz Biotechnology, sc-25778 |
ß-actin (C4) | 45 kDa | Mouse | 1:4000 | Santa Cruz Biotechnology, sc-47778 |
Lamin B1 | 66–70 kDa | Mouse | 1:4000 | ProteinTech 66095-1-Ig |
α-Rabbit | - | Goat | | Advansta, R05071 500 |
α-Mouse | - | Goat | | Advansta, R05072 500 |
α-Rat | - | Goat | | Advansta, R05075 500 |
Antibodies for Immunofluorescence Staining |
LAMP1 (CD107a) | 110 kDa | Mouse | 1:500 | BD Pharmingen, 555798 |
Cathepsin D | 28–45 kDa | Goat | 1:500 | R&D Systems, AF1014 |
LAMP2 (CD107b) | 120 kDa | Mouse | 1:150 | BD Pharmingen, 555803 |
TF-EB | 65–70 kDa | Rabbit | 1:200 | Cell Signaling Technologies, 4240 |
TF-E3 | 72–89 kDa | Rabbit | 1:250 | Cell Signaling Technologies, 14779 |
G3BP1 | 68 kDa | Mouse | 1:200 | Santa Cruz Biotechnology, sc-81940 |
Donkey α Mouse (488 nm) | - | Donkey | 1:250 | Life Technologies, A21202 |
Goat α Mouse (647 nm) | - | Goat | 1:250 | Invitrogen, A28181 |
Donkey α Rabbit (568 nm) | - | Donkey | 1:250 | Life Technologies, A10042 |
Donkey α Goat (647 nm) | - | Donkey | 1:250 | Life Technologies, A21447 |
Rabbit α Mouse IgG (bridging antibody) | - | Rabbit | 1:200 | Rockland, 610–4120 |
2.9 Proliferation and viability assays
Cells (1x104 cells/well) were seeded in complete medium and allowed to attach overnight. The following day, the cells were washed with PBS and then incubated for 48 h in either complete or nutrient-depleted medium and collected by trypsinization. For the Count and Viability Assay (Muse), cell suspensions were diluted 20-fold for LoVo and HCT-116 cells and 2-fold for T84 and Caco-2 cells using the Count & Viability Reagent (Luminex). The diluted cell suspension was incubated at room temperature for 5 min, and the stained cells were analyzed using a Muse Cell Analyzer (Luminex). For the Trypan Blue Exclusion Assay, the cells were resuspended in 1 mL of complete medium, mixed with 0.5% Trypan Blue solution at a 1:1 (v/v) ratio and incubated for 3 minutes at room temperature. The stained cells (10 µl) were placed on a hemocytometer, and unstained (viable) and stained (nonviable) cells were counted separately under a binocular light microscope. The percentage of viable cells was determined as a ratio of the viable cells to the total number of cells and presented as a percentage. For the MTT assay, 10,000 cells were plated in 96-well plates, and the appropriate treatments were carried out. Once the treatment duration was completed, the drug- or vehicle-containing cell culture medium was aspirated, and 100 µL of a 1.2 mM MTT solution was added to each well. After 4 hours of incubation, 100 µL of 1% SDS-0.01 M HCl solution was added to the MTT-added wells and incubated for another 18 hours at 37°C. A Multiskan-GO microplate reader (Thermo Fisher Scientific) was used to measure the absorbance at 570 nm. To determine any synergism between chemotherapeutic agents and bafilomycin A1 treatment, the percent viability data from the MTT assays were fed to the SynergyFinder 3.0 web tool using default settings.22 For the colony formation assay, T84 cells were seeded in 6-well plates to 50% confluency and incubated in nutrient-depleted or nutrient-rich medium for 48 hours. Upon completion of incubation, the cells were collected and counted, and 1000 viable cells per well were seeded into a 6-well plate in complete medium. The cells were incubated in a humidified incubator containing 5% CO2 at 37°C, and their medium was changed every 48 h. When the colonies were large enough to be seen with the naked eye (on the 10th day following seeding), the medium was removed, and the colonies were washed once with PBS. Next, the cells were fixed using 4% paraformaldehyde (PFA) for 15 minutes at room temperature, followed by staining in 0.5% crystal violet in methanol (Sigma Aldrich) for 20 minutes at room temperature on a rocker. The wells were gently washed with tap water to remove the excess crystal violet solution and left to air dry. The colonies were imaged and counted manually using the white tray of the ChemiDoc Imaging system and Image Lab software (Bio-Rad).
2.10 LysoTracker Staining
T84 and Caco-2 cells (1x104 cells/well) were seeded onto black, opaque, 96-well plates in complete medium and left for attachment overnight. The following day, the cells were washed with PBS and incubated in nutrient-rich or nutrient-depleted medium with and without 50 nM of the lysosomal V-ATPase inhibitor bafilomycin A1 for 48 h. Bafilomycin A1 served as a positive control since it causes alkalinization of lysosomes and is expected to decrease the LysoTracker signal.23 Upon completion of treatment, the cell medium from each well was aspirated, and the cells were washed with PBS once, followed by staining with 50 nM LysoTracker Red DND-99 (Thermo Fisher Scientific) and Hoechst 33342 Nuclear Stain (Enzo) in complete medium for 30 min at 37°C. The Hoechst 33342 nuclear stain served as an internal control to normalize the LysoTracker signal based on cell number. At the end of the incubation, the cells were washed twice with PBS, and the plate was read at excitation/emission 577 nm/590 nm for LysoTracker and excitation/emission 350 nm/461 nm for Hoechst 33342 using the SpectraMax iD3 multimode microplate reader (Molecular Devices).
2.11 Imaging Studies
2.11.1 Immunofluorescence (IF) Imaging
T84, HEK293 or HeLa cells (6x104 cells/well) were seeded onto sterile, 12–15 mm glass cover slips placed in 12-well plates in complete medium and left for attachment overnight. The following day, the cells were washed with PBS, and the treatments indicated in the figure legends were applied for 24 h. The next day, half-strength fixation was first carried out by adding 4% formaldehyde (FA, Sigma‒Aldrich) in 0.1 M phosphate buffer (PB) on top of each well in the same volume as the existing culture medium. The samples were incubated for 15 min at room temperature, and the fixation solution medium mixture was aspirated. Next, 500 µL of 4% FA was added to each well for full-strength fixation. The cells were incubated at room temperature for 2 h, after which the 4% FA was replaced with 1% FA, and the plates were stored at 4°C until use. The 4% FA was prepared freshly in PBS using a 16% stock solution of FA.
To perform IF staining, first, the coverslips were removed from the 12-well plate, and the fixed cells were washed twice with PBS. Next, the cells were permeabilized with 1% Triton X-100 for 10 min. Following a quenching step (to quench free aldehydes) with 0.15% glycine solution for 10 min, blocking was carried out for 10 min using 1% BSA. The cells were incubated with primary antibodies (Table 4) diluted in 1% BSA in PBS for 1 hour. Next, the cells were washed with PBS three times and incubated with the secondary antibody in 1% BSA for 30 min followed by washing. The coverslips were mounted on microscope slides using Prolong Diamond with DAPI (Thermo Fisher) and dried overnight. All steps of IF staining were carried out at room temperature (RT). The coverslips were visualized with the THUNDER Live-cell Imaging System (Leica) with a 100X, 1.47-NA oil objective. The number of cells imaged is indicated in the figure legends for each experiment separately. For the experiments in which Rab7a was silenced, HeLa cells were plated on sterile coverslips in a 12-well plate and allowed to attach overnight. The next day, the cells were transfected with siRNA using the same protocol as described for western blotting. After 24 h of transfection, the cells were fixed as described above and imaged using a Nikon Eclipse 55i fluorescence microscope using a 100X, 1.47-NA oil objective. All samples were imaged at RT, and image analyses were carried out using Fiji. Colocalization analysis was carried out with the Coloc2 plugin of Fiji.
2.11.2 Immunogold Electron Microscopy (EM) Imaging
The immuno-EM experiments were performed following the Tokuyasu method as reported previously.24 T84 cells were seeded in T25 flasks in complete medium and left for attachment overnight. The following day, the cells were washed with PBS, and the indicated treatments (nutrient-rich or -depleted medium containing 100 nM bafilomycin A1) were applied for 24 h. The next day, half-strength fixation was carried out by adding 4% freshly prepared formaldehyde (FA) into each flask in equal volume to the existing medium. After 15 min of incubation at room temperature, the fixation solution-medium mix was aspirated, and 1 mL of 4% FA was added to each well for full-strength fixation. The cells were further fixed at room temperature for 2 h. Once the fixation was completed, the fixative was washed off, and the sample was quenched with PBS containing 0.15% glycine. The cells were scraped from the flask and collected in PBS containing 1% gelatin. Next, the cells were centrifuged at 6000 x g at room temperature for 1 min, 1% gelatin was replaced by 12% gelatin at 37°C, and the cells were centrifuged at 6000 x g at room temperature for 1 min again. The pellets were placed on ice for freezing, cut into smaller blocks, and infused with 2.3 M sucrose overnight at 4°C. The smaller blocks were mounted on pins and stored in liquid nitrogen.
The gelatin-embedded cells were cryosectioned into either 250-nm semithickness or 90-nm thin sections at − 100°C on a diamond knife (Diatome) in an Ultracut Cryomicrotome (Leica). The sections were picked and deposited on formvar- and carbon-coated grids using 2.3 M sucrose and 1.8% methylcellulose (MC) mixed at a ratio of 1:1. The sections on grids were incubated in PBS at 37°C for ∼30 min to remove the gelatin, 2.3 M sucrose, and 1.8% MC mixture. After washing 3 times with PBS for 10 min and blocking with 1% BSA solution for 10 min, the sections were labelled using primary antibodies (Table 4) followed by incubation with bridging antibodies. The grids were then incubated with Protein A conjugated to 10–15-nm gold particles (Cell Microscopy Core, UMC Utrecht). The grids were poststained for 5 min using 2% uranium acetate (UA), pH 7.0, followed by embedding in a UA/MC mixture, pH 4.0 (2% MC/4%UA in a 9:1 ratio), for 10 min at 4°C. Imaging was carried out on a Tecnai T12 TEM (Thermo Fischer).
2.12 Chorioallantoic membrane (CAM) assay
Fertilized Leghorn specific-pathogen-free eggs were purchased from either VALO BioMedia (LSL Rhein-Main, Germany) for the data shown in Fig. 1, C and D or from Tavukçuluk Araştırma Enstitüsü (Ankara, Türkiye) for the data shown in Fig. 7D and Supplementary Fig. 4, A and C. The eggs were delivered at an ambient temperature of 12°C. The eggs were cleaned with tissue paper wetted with distilled water before putting them into a dedicated incubator at 37°C with 60–70% humidity for 7 days. On day 7, a tiny hole was generated with sterilized tweezers on the more rounded pole. On day 8, a window of approximately 1 cm was opened around the hole. A drop of sterile PBS was placed on the eggshell membrane, the CAM was pinched to allow the PBS to flow, and the eggshell membrane was removed. A piece of silk tape was used to seal the window, and the eggs were left in the incubator overnight. The next day, 20 µl of T84 cell suspension containing 1x106 viable cells was mixed with 20 µl of Matrigel (Corning). The cell suspension embedded in Matrigel was incubated for 10 minutes at room temperature followed by 1 h at 37°C and then placed on the CAM of the developing embryos. The eggs were placed in an incubator at 37°C with 60–70% humidity and incubated for another 5 days. Two independent sets of CAM assays were performed to determine the effect of nutrient depletion and to determine the effect of 5-FU in combination with bafilomycin A1 on tumor volume and proliferative capacity. In the CAM assays where the effect of nutrient depletion on microtumor volume and proliferative capacity were determined, 6 microtumors were harvested, and all were processed for Ki-67 staining. In the CAM assays where the combination of 5-FU and Bafilomycin A1 was tested, 6 microtumors were harvested, 4 microtumors were processed for Ki-67 staining and 2 microtumors were stored for future experiments. The harvested microtumors were measured in terms of height, width, and length for tumor volume calculation and then processed for immunohistochemistry (see below). Tumor volume was determined as pellet length x width x height × 0.52.25 Following the harvest of microtumors, the embryo was sacrificed immediately by decapitation with sharp scissors. The CAM assay is considered by the European Union as a non-animal experiment until hatching and therefore contributes to the 3R principles and does not require any approval from an Ethical Committee.26
2.13 Immunohistochemistry Staining
A drop of 0.2% eosin solution was used to stain microtumors collected from the CAM assay, and microtumors were gently wrapped in tissue 3x3 cm paper, placed into tissue embedding and processing cassettes, and then placed into 4% FA in PBS overnight fixation at room temperature. The next day, the cassettes were processed using routine tissue processing and paraffin embedding protocols. Then, 1–3 µm-thick sections were obtained from paraffin-embedded microtumors, processed for deparaffinization by incubation at 75°C for 40 min followed by two washes with xylene for 10 min each followed by rehydration in decreasing concentrations of ethyl alcohol (96%, 90%, and 70%, respectively) and gentle placement on microscopic slides. The sections were stained with hematoxylin–eosin for histological assessment of the sample, after which additional sections from the same tumors were immunohistochemically stained for Ki67. Routine protocols were applied for hematoxylin-eosin and immunohistochemistry staining for Ki67 (Dilution 1:100, Dako/Agilent, cat# M7240). Immunohistochemistry was performed on a Ventana BenchMark Ultra automated instrument (Ventana Medical Systems, Inc., Oro Valley, AZ, USA) in combination with the UltraView Universal DAB Detection Kit (Roche, Basel, Switzerland) according to the manufacturer’s recommendations.
2.14 Drug Correlation Analyses
Raw read counts of colon cancer cell lines (n = 48) were downloaded from the CCLE portal (https://portals.broadinstitute.org/ccle/data) and normalized with the DESeq2 package. The VST-transformed read counts for the genes of interest (e.g., RAB7A) were extracted and used for correlation analysis with drug sensitivity data. Drug sensitivity values [area under the curve (AUC)] for 550 compounds were downloaded from the National Cancer Institute database (https://ocg.cancer.gov/ctd2-data-project/broad-institute-screeningdependencies-cancer-cell-lines-using-small-molecules-0) and correlated with gene expression. The most significant correlation coefficient values (Pearson, p < 0.05) were identified. Correlation coefficients of gene expression (RAB7A, LAMP1, RAB5 and SNAP23) with drug sensitivity of colon cancer cell lines towards all 550 compounds in shown in Supplementary Table 2.
2.15 Statistical Analysis
Each experiment contained at least 2–3 biological replicates, each having at least two technical replicates. For data analysis, GraphPad Prism 6 (GraphPad Software Inc.) was used. To evaluate significance, one-way ANOVA or Student's t test was used. Statistical significance was defined as a p value of less than 0.05.