Registered report: Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of 50 papers in the field of cancer biology published between 2010 and 2012. This Registered Report describes the proposed replication plan of key experiments from ‘Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion’ by Straussman and colleagues, published in Nature in 2012 (Straussman et al., 2012). The key experiments being replicated in this study are from Figure 2A, C, and D (and Supplemental Figure 11) and Figure 4C (and Supplemental Figure 19) (Straussman et al., 2012). Figure 2 demonstrates resistance to drug sensitivity conferred by co-culture with some stromal cell lines and identifies the secreted factor responsible as HGF. In Figure 4, Straussman and colleagues show that blocking the HGF receptor MET abrogates HGF’s rescue of drug sensitivity. The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published by eLife. DOI: http://dx.doi.org/10.7554/eLife.04034.001


Introduction
Resistance to oncoprotein-targeted chemotherapy is a common occurrence during cancer treatment and identifying the mechanisms of resistance is important in improving treatment options. Specifically, BRAFmutant melanomas, which show an initial response to RAF inhibitors, usually become resistant to the therapy (Nickoloff and Vande Woude, 2012). The identification of stroma-mediated resistance in BRAFmutant melanomas, through the secretion of hepatocyte growth factor (HGF), therefore indicates a potential therapeutic strategy through combination treatment of RAF inhibitors and inhibition of the HGF activated pathway (Straussman et al., 2012). This report is the first to identify paracrine HGF as a potential mechanism for the development of drug resistance (Ghiso and Giordano, 2013;Glaire et al., 2012).
In Figure 2A of their paper, Straussman and colleagues tested the effect of fibroblast-conditioned medium on the proliferation of BRAF-mutant melanoma cells grown in the presence of the BRAF inhibitor PLX4720. Using a cell proliferation assay, they reported that fibroblast-conditioned medium rescued BRAF-mutant melanoma cells from PLX4720 sensitivity, which indicated that a secreted factor was involved. This was a key finding demonstrating that the stromal environment of the tumor cells could mediate their response to drug treatment. This experiment will be replicated in Protocol 3.
Straussman and colleagues went on to identify the secreted factor responsible for acquired drug resistance as HGF. In Figure 2C, they demonstrated that treating melanoma cell lines with PLX4720 in combination with increasing concentrations of exogenous HGF increased proliferation as compared to treatment with drug alone. This finding showed a similar effect to treatment with conditioned media from stromal cells that secrete HGF (see Figure 2A) and supported the hypothesis that HGF is the growth factor responsible for rescuing melanoma cells from drug sensitivity. This experiment will be replicated in Protocol 4. Straussman and colleagues demonstrated that the HGF-mediated rescue of melanoma cells from drug sensitivity was mediated through HGF's cognate receptor tyrosine kinase MET by treating melanoma cell lines co-cultured with stromal cell lines in the presence of PLX4720 with the MET inhibitor crizotinib, as shown in Figure 2D and Supplemental Figure 11. Treatment with crizotinib reduced the increase in proliferation due to co-culture with an HGF-secreting stromal cell line. This experiment provided further support for the hypothesis that HGF was responsible for rescue from drug sensitivity and also provided evidence that that rescue was MET dependent. This experiment is replicated in Protocol 5. Lastly, Straussman and colleagues reported sustained activation of both ERK and AKT in HGF-treated melanoma cells during BRAF inhibition and to a lesser extent MEK inhibition, as shown in Figure 4C and Supplemental Figure 19 by Western blot. This confirmed activation of pro-survival pathways in response to HGF treatment even in the presence of PLX4720. These experiments are replicated in Protocol 6.
To date, a direct replication has been reported; Lezcano and colleagues (Lezcano et al., 2014) published a replication of Figure 3 of Straussman et al. Nature 2013, wherein Straussman and colleagues evaluated HGF expression in patient-derived primary melanoma samples and observed a negative correlation between expression of HGF and response to therapy (Straussman et al., 2012). While Lezcano and colleagues' replication also detected the presence of HGF in human melanoma tumor cells and stromal cells with increased expression at disease progression, they did not identify a statistically significant correlation between HGF expression and clinical outcome (Lezcano et al., 2014). While both of the studies come to different conclusions about the association of stromal HGF and clinical outcome, the 95% confidence intervals of the standardized measure of the effect (Cohen's d) for each study substantially overlap. A study published around the same time as the work of Straussman and colleagues supports the negative association between HGF and clinical response to RAF inhibitor treatments through an analysis of HGF levels in patient plasma samples (Wilson et al., 2012).
In other systems, additional labs have observed a similar role for HGF in acquired drug resistance. Caenepeel and colleagues reported that HGF rescued melanoma cell lines, notably SK-MEL-5, from BRAF or MEK inhibition using vemurafenib (an analogue of PLX4720) or PD0325901, respectively, and the rescue was attenuated by MET inhibition (Caenepeel et al., 2013). Nakagawa and colleagues observed that tumor-secreted (not stromal secreted) HGF could induce resistance to the VEGFR inhibitor lenvatinib, and that this resistance could be overcome by co-treatment with golvatinib, a MET inhibitor (Nakagawa et al., 2014). Etnyre and colleagues reported that c-MET and BRAF inhibitors had synergistic inhibitory effects when exposed in combination to melanoma cell lines (Etnyre et al., 2013). Casbas-Hernandez and colleagues co-cultured MCF10 cells with immortalized mammoplasty derived fibroblasts and observed a correlation between the levels of fibroblast-secreted HGF and the differentiation of the MCF10 cells towards a ductal carcinoma phenotype. They also observed a correlation between HGF expression and the more invasive basal-like tumors as opposed to the less invasive luminal tumors (Casbas-Hernandez et al., 2013). HGF is also being evaluated as a potential biomarker to indicate potential treatment choices (Penuel et al., 2013;Xie et al., 2013).

Materials and methods
Unless otherwise noted, all protocol information was derived from the original paper, references from the original paper, or information obtained directly from the authors.

Materials and reagents
• Reagents that are different from ones originally used are noted with an asterisk (*). Procedure 1. Seed 4 wells of a 384-well clear-bottom plate with 8000 cells/well all the way to 31.25 cells/well (serial 1:2 dilutions) with pLex-TRC206 SK-MEL-5 cells in 60 µl per well using phenol red free medium using an automated workstation.
Note: all cells will be sent for mycoplasma testing and STR profiling. Note: ensure at least 85% of SK-MEL-5 cells are GFP-positive before start of the experiment. Cells can be enriched using FACS or puromycin (0.5-2 µg/ml), however do not grow cells under antibiotic selection on a regular basis.
A. Total wells seeded = 36 B. Medium for assay: phenol red free DMEM supplemented with 1 mM sodium pyruvate, 10% FBS, and 1X Pen-Strep-Glut. C. Fill wells with 60 µl/well of clear media in at least 2 rows and 2 columns around wells that are being included in the experiment.
2. The next day after seeding, read GFP fluorescence (Synergy HT Microplate Reader).
A. Subtract the average reading from media-only wells from the wells with cells.

Deliverables
• Data to be collected:

Provisions for quality control
This protocol will ensure that the replicating lab's plate reader is comparable to the original lab's plate reader.
• A lab from the Science Exchange network with extensive experience in conducting cell viability assays will perform these experiments. • All cells will be sent for STR profiling to confirm identity and mycoplasma testing to confirm the lack of mycoplasma contamination. • SK-MEL-5 cells will be confirmed to have at least 85% of the cells GFP-positive before the start of the experiment.
Protocol 2: determining the detection variability of the replicating lab's plate reader This is a general protocol that determines the variability in detection of the plate reader. Because the plate reader in use by the replicating lab is different than the plate reader used in the original study, we are determining what the variability of detection is for the replicating lab's plate reader.

2000 cells/well x 384 replicates
• Experiment will be done a total of once.

Materials and reagents
• Reagents that are different from ones originally used are noted with an asterisk (*). Note: all cells will be sent for mycoplasma testing and STR profiling. Note: ensure at least 85% of SK-MEL-5 cells are GFP-positive before start of the experiment. Cells can be enriched using FACS or antibiotics, however do not grow cells under antibiotic selection on a regular basis.

Reagent
A. Medium for assay: phenol red free DMEM supplemented with 1 mM sodium pyruvate, 10% FBS, and 1× Pen-Strep-Glut. B. Fill wells with 60 µl/well of clear media in at least 2 rows and 2 columns around wells that are being included in the experiment.
2. The next day after seeding, read GFP fluorescence (Synergy HT Microplate Reader).
A. Subtract the average reading from media only wells from the wells with cells.

Deliverables
• Data to be collected:

Provisions for quality control
This protocol will ensure that the replicating lab's plate reader is comparable to the original lab's plate reader.
• A lab from the Science Exchange network with extensive experience in conducting cell viability assays will perform these experiments. • All cells will be sent for STR profiling to confirm identity and mycoplasma testing to confirm the lack of mycoplasma contamination. • SK-MEL-5 cells will be confirmed to have at least 85% of the cells GFP-positive before the start of the experiment.

Protocol 3: co-culture proliferation assay
This protocol outlines how to culture melanoma cell lines with conditioned medium from three stromal cell lines with or without the RAF inhibitor PLX4720 to analyze cell proliferation rates, as is described in Figure 2A.

Sampling
• Experiment to be repeated a total of 4 times for a minimum power of 81%.

See Power calculations section for details
• Each experiment has six conditions to be run in quadruplicate per experiment: 1. SK-MEL-5 untreated control [additional control] 2. SK-MEL-5 vehicle (DMSO) control 3. SK-MEL-5 treated with 2 µM PLX4720 and with unconditioned medium 4. SK-MEL-5 treated with 2 µM PLX4720 and with conditioned medium from CCD-1090Sk cells that do not secrete HGF 5. SK-MEL-5 treated with 2 µM PLX4720 and with conditioned medium from PC60163A1 cells that do secrete HGF 6. SK-MEL-5 treated with 2 µM PLX4720 and with conditioned medium from LL 86 cells that do secrete HGF

Materials and reagents
• Reagents that are different from ones originally used are noted with an asterisk (*).

Procedure
1. Prepare Pre-Conditioned Medium (PCM); fresh PCM must be prepared the same day it is used in the treatment of SK-MEL-5 cells; this step is repeated three times to ensure fresh PCM is available on the needed day: A. Three days before the PCM is needed, seed 3 × 10 cm tissue culture plates with 0.5x10 6 LL 86 cells each, 3 × 10 cm tissue culture plates with 1x10 6 PC60163A1 cells each, and 3 × 10 cm tissue culture plates with 2 × 10 6 CCD-1090Sk cells each (9 plates total) in 10 ml of phenol red free medium each and grow for 3 days. B. 3 days after seeding, collect the medium from each cell line using the plate closest to 80-90% confluent.

Reagent
Type Registered report C. Filter through 0.45 µm syringe filter with a 10 ml syringe and dilute filtered PCM 1:1 in fresh phenol red free medium. Total volume = 20 ml.
i. Use the same day.
ii. Do not dilute for day 0 of treatment (these wells will already have 20 µl of media in them).
2. On day 0, seed 120 wells of a 384-well clear-bottom plate with 1900 pLex-TRC206 SK-MEL-5 cells in 20 µl per well using phenol red free medium using an automated workstation. Note: 1. All cells will be sent for mycoplasma testing and STR profiling.
2. Ensure at least 85% of SK-MEL-5 cells are GFP-positive before start of the experiment. Cells can be enriched using FACS or antibiotics, however do not grow cells under antibiotic selection on a regular basis. 3. Do not exceed a rate of 5-10 µl/s and do not let the tip end closer than 1 mm to the well bottom.
A. Fill wells with 50 µl/well of media in at least 2 rows and 2 columns around wells that are being included in the experiment.
B. To wells in step A, add 20 µl of fresh undiluted PCM from appropriate stromal cells generated as described in step 1 (see Sampling section for Cohorts) or phenol red free medium alone (Cohort 1).
A. Subtract the average reading from media-only wells from the wells with cells.
4. After reading GFP fluorescence, refresh media and add drug using an automated workstation.
A. Change the medium for each cohort to 40 µl fresh diluted PCM from appropriate stromal cell lines generated as described in step 1 or phenol red free medium alone. B. Within each cohort, add 10 µl of 5X PLX4720, DMSO dilution, or 10 µl phenol red free medium to each appropriate well to bring the final volume per well up to 50 µl.
i. 5X PLX4720: make up stocks of 10 mM PLX4720 in DMSO, then dilute 1:1000 in media to make up 10 µM PLX4720. This is a 5× stock. ii. DMSO dilution: dilute 1 µl DMSO with 999 µl media. Add 10 µl of this mix to DMSO wells.
1. These dilutions in media prevent toxicity from excess DMSO.

On day 4 after seeding, read GFP fluorescence.
A. Subtract the average reading from media-only wells from the wells with cells.
6. After reading GFP fluorescence, change the medium in appropriate wells to 40 µl fresh diluted PCM from appropriate stromal cell lines generated as described in step 1 or phenol red free medium alone using an automated workstation.
A. Add 10 µl of 5X PLX4720, DMSO dilution, or 10 µl phenol red free medium to each appropriate well to bring the final volume per well up to 50 µl.
i. 5X PLX4720: make up stocks of 10 mM PLX4720 in DMSO, then dilute 1:1000 in media to make up 10 µM PLX4720. This is a 5× stock. ii. DMSO dilution: dilute 1 µl DMSO with 999 µl media. Add 10 µl of this mix to DMSO wells.
A. Subtract the average reading from media-only wells from the wells with cells.

Data analysis:
A. Remove background fluorescence by subtracting the average reading from media-only wells from the wells with cells for each plate reading. B. Subtract the readings of day 1 from the other plates (day 4 and day 7) for the same wells. C. Average the quadruplicates. D. Calculate the effect of PLX4720 in the presence or absence of conditioned media by normalizing the number of cells after 7 days of treatment (as measured by GFP fluorescence) to the number of cells present in the SK-MEL-5 vehicle control condition.
9. Repeat experiment independently three additional times.

Confirmatory analysis Plan
• Statistical analysis of the replication data: A. One-way ANOVA comparing the proliferation of PLX4720-treated cells cultured with unconditioned medium, CCD-1090Sk conditioned medium, LL 86 conditioned medium, or PC60163A1 conditioned medium.

Planned comparisons with the Bonferroni correction:
• Unconditioned medium to PC60163A1 conditioned medium • Unconditioned medium to LL 86 conditioned medium • CCD-1090Sk to PC60163A1 conditioned medium • CCD-1090Sk to LL 86 conditioned medium Meta-analysis of original and replication attempt effect sizes: A. Compare the effect sizes of the original data to the replication data and use a meta-analytic approach to combine the original and replication effects, which will be presented as a forest plot.
Known differences from the original study • The replication will only use one of the three melanoma cell lines used by the original authors, the SK-MEL-5 cell line. The replication will exclude SK-MEL-28 and G-361 cells. • The replication will include an additional control, untreated SK-MEL-5 cells in addition to the vehicle (DMSO) treated SK-MEL-5 cells used in the original study. • A Synergy HT Microplate Reader will be used instead of a Molecular Devices SpectraMax M5e Microplate Reader-both can detect GFP fluorescence and the Synergy HT Microplate Reader will be evaluated for range of detection (Protocol 1) and detection variability (Protocol 2) • A BD Pathway 435 Bioimager used instead of a Zeiss Axio Observer.Z1-both are fluorescence microscopes with high-throughput screening capabilities. • The replicating lab does not have a ViCell XR cell viability counter, and thus will seed a larger number of cells per well (1900 instead of 1700 cells/well).

Provisions for quality control
All data obtained from the experiment-raw data, data analysis, control data, and quality control datawill be made publicly available, either in the published manuscript or as an open access dataset available on the Open Science Framework (https://osf.io/p4lzc/).
• A lab from the Science Exchange network with extensive experience in conducting cell viability assays will perform these experiments. • All cells will be sent for STR profiling to confirm identity and mycoplasma testing to confirm the lack of mycoplasma contamination. • SK-MEL-5 cells will be confirmed to have at least 85% of the cells GFP-positive before the start of the experiment.

Protocol 4: recombinant HGF proliferation assay
This protocol assesses changes in proliferation when melanoma cells are treated with the RAF inhibitor PLK4720 with or without HGF, as is described in Figure 2C. The cells are also treated with a MEK inhibitor, PD184352.

Sampling
• Experiment to be repeated a total of three times for a final power of 99%.

Materials and reagents
• Reagents that are different from the ones originally used are noted with an asterisk (*).

Reagent
Type Manufacturer Catalog # Comments 1. All cells will be sent for mycoplasma testing and STR profiling. 2. Ensure at least 85% of SK-MEL-5 cells are GFP-positive before start of the experiment. Cells can be enriched using FACS or antibiotics, however do not grow cells under antibiotic selection on a regular basis. 3. Do not exceed a rate of 5-10 µl/s and do not let the tip end closer than 1 mm to the well bottom. A. Fill wells with 60 µl/well of clear media in at least 2 rows and 2 columns around wells that are being included in the experiment. B. Medium of all cell lines for assay: phenol red free DMEM supplemented with 1 mM sodium pyruvate, 10% FBS, and 1× Pen-Strep-Glut.
A. Subtract the average reading from media-only wells from the wells with cells.
3. After reading GFP fluorescence, add to the appropriate wells 10 µl 6X HGF or phenol red free medium alone. Then add to the appropriate wells the following: 10 µl 6X PLX4720, 10 µl 6X PD184352, 10 µl DMSO dilution, or 10 µl phenol red free medium alone.
A. 6X HGF: make up stocks of 100 μg/ml HGF, then dilute accordingly to make 6X working concentrations of each required HGF dilution. B. 6X PLX4720: make up stocks of 12 mM PLX4720 in DMSO, then dilute 1:1000 in media to make up 12 μM PLX4720 for use at 6X for the assay to avoid excessive DMSO toxicity. C. 6X PD184352: make up stocks of 6 mM PD184352 in DMSO, then dilute 1:1000 in media to make up 6 μM PD184352 for use at 6X for the assay to avoid excessive DMSO toxicity. D. DMSO dilution: dilute 1 µl DMSO with 999 µl media. Add 10 µl of this mix to DMSO dilution wells.
A. These media dilutions are to avoid toxicity from excessive DMSO.
A. Subtract the average reading from media-only wells from the wells with cells.

5.
After reading GFP fluorescence, change the medium in all wells to 40 µl fresh phenol red free medium using an automated workstation. Then add to the appropriate wells 10 µl 6X HGF or phenol red free medium alone. Then add to the appropriate wells the following: 10 µl 6X PLX4720, 10 µl 6X PD184352, 10 µl DMSO dilution, or 10 µl phenol red free medium alone.
A. 6X HGF: make up stocks of 100 μg/ml HGF, then dilute accordingly to make 6X working concentrations of each required HGF dilution. B. 6X PLX4720: make up stocks of 12 mM PLX4720 in DMSO, then dilute 1:1000 in media to make up 12 μM PLX4720 for use at 6X for the assay to avoid excessive DMSO toxicity. C. 6X PD184352: make up stocks of 6 mM PD184352 in DMSO, then dilute 1:1000 in media to make up 6 μM PD184352 for use at 6X for the assay to avoid excessive DMSO toxicity. D. DMSO dilution: dilute 1 µl DMSO with 999 µl media. Add 10 µl of this mix to DMSO dilution wells. A. These media dilutions are to avoid toxicity from excessive DMSO.
6. On day 7 after seeding, read GFP fluorescence and document bright-field and GFP images (BD, Pathway 435 Bioimager). A. Subtract the average reading from media-only wells from the wells with cells.

Data analysis:
A. Remove background fluorescence by subtracting the average reading from media-only wells from the wells with cells for each plate reading. B. Subtract the readings of day 1 from the other plates (day 4 and day 7) for the same wells. C. Average the quadruplicates. D. Calculate the effect of PLX4720 and PD184352 in the presence or absence of HGF by normalizing the number of cells after 7 days of treatment (as measured by GFP fluorescence) to the number of cells present in the SK-MEL-5 vehicle control condition.
8. Repeat the experiment independently two additional times.
3. Fluorescent and bright-field micrographs of cells from day 7. 4. Bar chart of relative proliferation as a % of untreated control for all conditions. (Use data from Day 7 -Day 1 background) (Compare to Figure 2C) 5. A semi-logarithmic graph of proliferation (log) vs time (linear) over 3 time points after seeding.

Confirmatory analysis Plan
• Statistical Analysis: 1. Compare the proliferation rate of PLX4720-treated cells treated with 0, 6.25, 12.5, 25, or 50 ng/ml HGF. Also compare each HGF cohort to the proliferation rate of vehicle-treated and untreated cells.
A. One-way ANOVA 2. Compare the proliferation rate of PD184352-treated cells treated with 0, 6.25, 12.5, 25, or 50 ng/ml HGF. Also compare each HGF cohort to the proliferation rate of vehicle-treated and untreated cells.
A. One-way ANOVA • Meta-analysis of original and replication attempt effect sizes: 1. Compare the effect sizes of the original data to the replication data, using a meta-analytic approach to combine the original and replication effects which will be presented as a forest plot.

Known differences from the original study
• The replication will only use one of the three melanoma cell lines used by the original authors, the SK-MEL-5 cell line. The replication will exclude SK-MEL-28 and G-361 cells.

• The replication will include an additional control, untreated SK-MEL-5 cells in addition to the vehicle (DMSO) treated SK-MEL-5 cells used in the original study. • A Synergy HT Microplate Reader used instead of a Molecular Devices SpectraMax M5e Microplate
Reader-both can detect GFP fluorescence and the Synergy HT Microplate Reader will be evaluated for range of detection (Protocol 1) and detection variability (Protocol 2) • A BD Pathway 435 Bioimager used instead of a Zeiss Axio Observer.Z1-both are fluorescence microscopes with high-throughput screening capabilities. • The replicating lab does not have a ViCell XR cell viability counter and thus will seed a larger number of cells per well (2800 instead of 2500 cells/well).

Provisions for quality control
All data obtained from the experiment-raw data, data analysis, control data, and quality control data-will be made publicly available, either in the published manuscript or as an open access dataset available on the Open Science Framework (https://osf.io/p4lzc/).
• A lab from the Science Exchange network with extensive experience in conducting cell viability assays will perform these experiments. • All cells will be sent for STR profiling to confirm identity and mycoplasma testing to confirm the lack of mycoplasma contamination.
• SK-MEL-5 cells will be confirmed to have at least 85% of the cells GFP-positive before the start of the experiment.

Protocol 5: inhibitor proliferation assay
This experiment confirms that the rescue from drug sensitivity is due to HGF signaling by co-treating cells with crizotinib, an inhibitor of MET, the receptor tyrosine kinase for HGF, as seen in Figure 2D and Supplemental Figure 11.

Sampling
• Run the experiment six times in total for a minimum power of 80%.

See Power calculations section for details
• Each experiment has 10 cohorts:
2. The cohorts are treated with the following drugs: •  Note: 1. All cells will be sent for mycoplasma testing and STR profiling.
2. Ensure at least 85% of SK-MEL-5 cells are GFP-positive before the start of the experiment. Cells can be enriched using FACS or antibiotics, however do not grow cells under antibiotic selection on a regular basis. 3. Do not exceed a rate of 5-10 µl/s and do not let the tip end closer than 1 mm to the well bottom A. Total wells seeded: 120 B. Fill wells with 60 µl/well of clear media in at least 2 rows and 2 columns around wells that are being included in the experiment. C. Medium of all cell lines for assay: phenol red free DMEM supplemented with 1 mM sodium pyruvate, 10% FBS, and 1X Pen-Strep-Glut.

In wells from
Step 1, seed 1900 pLex-TRC206 SK-MEL-5 cells in 20 µl phenol red free medium per well using an automated workstation. 3. On day 1 after seeding, read GFP fluorescence (Synergy HT Microplate Reader).
A. Subtract the average reading from media-only wells from the wells with cells.

Add appropriate drugs to each well (final volume = 60 µl).
A. Formulation of drug stock solutions: i. 6X PLX4720: make up stocks of 12 mM PLX4720 in DMSO, then dilute 1:1000 in media to make up 12 µM PLX4720 for use at 6× for the assay to avoid excessive DMSO toxicity. ii. 6X PD184352: make up stocks of 6 mM PD184352 in DMSO, then dilute 1:1000 in media to make up 6 µM PD184352 for use at 6× for the assay to avoid excessive DMSO toxicity. iii. 6X crizotinib: make up stocks of 1.2 mM crizotinib in DMSO, then dilute 1:1000 in media to make up 1.2 µM PD184352 for use at 6× for the assay to avoid excessive DMSO toxicity. iv. 6X PHA-665752: make up stocks of 1.2 mM PHA-665752 in DMSO, then dilute 1:1000 in media to make up 1.2 µM PD184352 for use at 6× for the assay to avoid excessive DMSO toxicity. V. DMSO dilution: dilute DMSO 1:1000 in medium to avoid excessive DMSO toxicity.
Known differences from the original study • Supplemental Figure 11 tests co-culture of SK-MEL-5 cells with 9 stromal cell lines. We have chosen LL86 cells, which showed the largest rescue of proliferation, and CCD-1090Sk cells, which showed the least rescue. • Additional controls added by the replication team: 1. Treatment with PHA-665752 • In addition to inhibiting MET, crizotinib also targets ALK, ROS1, and RON. In order to confirm that the effects of crizotinib are due to targeting of MET, we will also use a more selective MET inhibitor, PHA-665752 (Cui, 2014;Parikh et al., 2014).
2. The replication will include an additional control, untreated SK-MEL-5 cells in addition to the vehicle (DMSO) treated SK-MEL-5 cells used in the original study.
• A Synergy HT Microplate Reader used instead of a Molecular Devices SpectraMax M5e Microplate Reader 1. Both can detect GFP fluorescence and the Synergy HT Microplate Reader will be evaluated for range of detection (Protocol 1) and detection variability (Protocol 2) • A BD Pathway 435 Bioimager used instead of a Zeiss Axio Observer.Z1 1. Both are fluorescence microscopes with high-throughput screening capabilities.
• The replicating lab does not have a ViCell XR cell viability counter and thus will seed a larger number of cells per well (1900 instead of 1700 cells/well).

Provisions for quality control
All data obtained from the experiment -raw data, data analysis, control data, and quality control data -will be made publicly available, either in the published manuscript or as an open access dataset available on the Open Science Framework (https://osf.io/p4lzc/).
• A lab from the Science Exchange network with extensive experience in conducting cell viability assays will perform these experiments. • All cells will be sent for STR profiling to confirm identity and mycoplasma testing to confirm the lack of mycoplasma contamination. • SK-MEL-5 cells will be confirmed to have at least 85% of the cells GFP-positive before the start of the experiment.

Protocol 6: inhibitor Western blot assay of ERK and AKT signaling
This experiment assesses the protein levels of various activated downstream pathway signaling component proteins in the presence or absence of HGF and drugs, as seen in Figure 4C and Supplemental Figure 19.

Sampling
• Repeat the experiment six times in total for a minimum power of 85%.

See Power calculations section for details
• Each experiment contains seven conditions:  Table 6. Continued on next page Registered report Procedure 1. On day 0, plate 5 × 10 5 pLex-TRC206 SK-MEL-5 cells in 2 ml media per well for a total of 7 wells across 2 × 6-well plates. Note: 1. All cells will be sent for mycoplasma testing and STR profiling. 2. Ensure at least 85% of SK-MEL-5 cells are GFP-positive before start of the experiment. Cells can be enriched using FACS or antibiotics, however do not grow cells under antibiotic selection on a regular basis. A. Medium of all cell lines for assay: DMEM supplemented with 10% FBS and 1× Pen-Strep.
2. On day 1 add the appropriate additives to each well.
A. Formulation of stock solutions: Note: these dilutions are to avoid toxicity from excessive DMSO.
i. 1000X HGF: make a stock of 25 µg/ml HGF. ii. 1000X PLX4720: make a stock of 20 mM PLX4720 in DMSO, then dilute 1:10 in media to make a 2 mM PLX4720 working solution. iii. 1000X PD184352: make a stock of 20 mM PD184352 in DMSO, then dilute 1:10 in media to make a 2 mM working solution. iv. DMSO dilution: dilute DMSO 1:10 in medium.
3. 24 hr after drug treatment, prepare cells for lysis.
A. Quickly wash cells with ice-cold PBS and remove excess PBS. B. Add 0.5 ml or less of ice-cold lysis buffer to wells on ice.
C. Scrape cells off dish with cell scraper. D. Collect cells in a 1.5 ml centrifuge tube on ice.

Confirmatory analysis plan
• Statistical analysis of replication data: 1. Two-way ANOVA comparing the relative phopho-AKT band intensities of cells treated with vehicle, PLX4720, or PD184352 in the presence or absence of HGF.
A. Planned comparisons with the Bonferroni correction: • PLX4720-treated cells in the absence of HGF compared to PLX4720-treated cells in the presence of HGF.
2. Two-way ANOVA comparing the relative phopho-ERK band intensities of cells treated with vehicle, PLX4720, or PD184352 in the presence or absence of HGF.
A. Planned comparisons with the Bonferroni correction: • PLX4720-treated cells in the absence of HGF compared to PLX4720-treated cells in the presence of HGF.
3. Two-way ANOVA comparing the relative phopho-MET (Tyr1349) band intensities of cells treated with vehicle, PLX4720, or PD184352 in the presence or absence of HGF.
A. Planned comparisons with the Bonferroni correction: • Cells treated in the absence of HGF and treated with vehicle, PLX4720, or PD184352 compared to cells treated in the presence of HGF and treated with vehicle, PLX4720, or PD184352.
• Meta-analysis of original and replication attempt effect sizes: 1. Compare the effect sizes of the original data to the replication data and use a meta-analytic approach to combine the original and replication effects, which will be presented as a forest plot.
Known differences from the original study • Provider lab transfer protocol used instead of iBlot Gel Transfer Device (Invitrogen, IB1001) using Program 4-both are capable of transferring protein efficiently, and to determine completeness of the transfer the gel may be stained (Step 8). • The replication will include an additional control, untreated SK-MEL-5 cells in addition to the vehicle (DMSO) treated SK-MEL-5 cells used in the original study. • The replication will not include the pMet Tyr1234/5, RAF1, and pRAF1 antibodies included in the original study.

Provisions for quality control
All data obtained from the experiment-raw data, data analysis, control data, and quality control data-will be made publicly available, either in the published manuscript or as an open access dataset available on the Open Science Framework (https://osf.io/p4lzc/).
• A lab from the Science Exchange network with extensive experience in conducting cell viability assays and performing Western blots will perform these experiments. • All cells will be sent for STR profiling to confirm identity and mycoplasma testing to confirm the lack of mycoplasma contamination. • SK-MEL-5 cells will be confirmed to have at least 85% of the cells GFP-positive before the start of the experiment.

Power calculations
All calculations are determined in order to reach at least 80% power.

Protocol 1
No power calculations required.

Protocol 2
No power calculations required.

Protocol 3
Summary of original data: Note: original data values were shared by authors.
• Standard deviation was calculated using the formula, SD = SEM*(SQRT n)

Test family
• ANOVA: fixed effects, omnibus, one-way, alpha error = 0.05 • Power calculations were performed from effects reported in the original study using G*Power software (version 3.1.7) (Faul et al., 2007). • ANOVA F statistic calculated with Graphpad Prism 6.0 • Partial η 2 calculated from Lakens (2013) Power calculations for replication Test family • Two tailed t-test; difference between two independent means Bonferroni's correction: alpha error = 0.0125.
• Calculations were performed from effects reported in the original study using G*Power software (version 3.1.7) (Faul et al., 2007). • Standard deviation was calculated using the formula, SD = SEM*(SQRT n)

Test family
• ANOVA: fixed effects, omnibus, one-way, alpha error = 0.05 • Power calculations were performed from effects reported in the original study using G*Power software (version 3.1.7) (Faul et al., 2007). • ANOVA F statistic calculated with Graphpad Prism 6.0 • Partial η 2 calculated from Lakens (2013) Power calculations for replication Summary of original data Note: original data values were shared by authors.
• Standard deviation was calculated using the formula, SD = SEM*(SQRT n)

Test family
• ANOVA: fixed effects, omnibus, one-way, alpha error = 0.05 • Power calculations were performed from effects reported in the original study using G*Power software (version 3.1.7) (Faul et al., 2007). • ANOVA F statistic calculated with Graphpad Prism 6.0 • Partial η 2 calculated from Lakens (2013 • Power calculations were performed from effects reported in the original study using G*Power software (version 3.1.7) (Faul et al., 2007 10,000 simulations were run using the summary data to randomly assign data values and the interaction F statistic was computed for a 3-way ANOVA between subjects design. The average F statistic was calculated and used in the power calculations.

Power calculations for replication
Test family • 2-way ANOVA between subjects: fixed effects, special, main effects, and interactions, alpha error = 0.05 • Power calculations were performed from effects reported in the original study using G*Power software (version 3.1.7) (Faul et al., 2007). • ANOVA F statistic calculated with Graphpad Prism 6.0 • Partial η 2 calculated from Lakens (2013) Power calculations for replication (BRAF/MEK inhibitor)

Test family
• Two tailed t-test; difference between two independent means, Bonferroni's correction: alpha error = 0.0125.
• Power calculations were performed for effects reported in the original study using G*Power software (version 3.1.7) (Faul et al., 2007).
Power calculations for replication (PLX4720 group)

Test family
• Two tailed t-test; difference between two independent means, Bonferroni's correction: alpha error = 0.025.
• Power calculations were performed for effects reported in the original study using G*Power software (version 3.1.7) (Faul et al., 2007).
Power calculations for replication (PD184352 group)

Protocol 6 Summary of original data
Note: numbers were estimated from bar chart in Supplemental Figure S19. Test family • 2-way ANOVA between subjects: fixed effects, special, main effects, and interactions, alpha error = 0.05 • Power calculations were performed from effects reported in the original study using G*Power software (version 3.1.7) (Faul et al., 2007). • ANOVA F statistic calculated with Graphpad Prism 6.0 • Partial η 2 calculated from Lakens (2013) Power calculations for replication

Test family
• Two tailed t-test; difference between two independent means, Bonferroni's correction: alpha error = 0.05.

Registered report
Test family • Two tailed t-test; difference between two independent means, Bonferroni's correction: alpha error = 0.05. o Note: calculations were performed for effects reported in the original study using G*Power software (version 3.1.7) (Faul et al., 2007).

Power calculations for replication (pERK group)
Test family • Two tailed t-test; difference between two independent means, Bonferroni's correction: alpha error = 0.05. o Note: calculations were performed for effects reported in the original study using G*Power software (version 3.1.7) (Faul et al., 2007).