Registered report: Biomechanical remodeling of the microenvironment by stromal caveolin-1 favors tumor invasion and metastasis.

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replicating selected results from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012 were selected on the basis of citations and Altimetric scores (Errington et al., 2014). This Registered report describes the proposed replication plan of key experiments from 'Biomechanical remodeling of the microenvironment by stromal caveolin-1 favors tumor invasion and metastasis' by Goetz and colleagues, published in Cell in 2011 (Goetz et al., 2011). The key experiments being replicated are those reported in Figures 7C (a-d), Supplemental Figure S2A, and Supplemental Figure S7C (a-c) (Goetz et al., 2011). In these experiments, which are a subset of all the experiments reported in the original publication, Goetz and colleagues show in a subcutaneous xenograft model that stromal caveolin-1 remodels the intratumoral microenvironment, which is correlated with increased metastasis formation. 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 in eLife.

Introduction

The importance of the tumor microenvironment in cancer progression is well established. Cancer-associated fibroblasts (CAFs) are one subset of cells found in the tumor stroma that help to regulate tumor progression and metastasis through several mechanisms such as remodeling of the extracellular matrix, secretion of growth factors and chemokines, and regulation of epithelial-to-mesenchymal transition (Kalluri and Zeisberg, 2006; Cirri and Chiarugi, 2011). Caveolin-1 (Cav1) is an essential component of caveolae and regulator of lipid raft formation that plays an important role in tumor progression (Goetz et al., 2008; Sotgia et al., 2012). However, the precise role of Cav1 in tumor progression appears to be unclear, with studies showing both increased and decreased expression in various types of cancer (Parton and del Pozo, 2013). One possible model is a general trend in which Cav1 appears to act as a tumor suppressor at early stages of cancer progression, but is up-regulated in several multidrug-resistant and metastatic cancer cell lines and human tumor specimens, positively correlating with tumor stage and grade in numerous cancer types (Shatz and Liscovitch, 2008). Part of this variability could stem from Cav1 expression in stromal rather than tumor cells. For instance, loss of Cav1 function in stromal cells of various organs leads to benign stromal lesions responsible for abnormal growth and differentiation of the epithelium and to dramatic reductions in life span (Yang et al., 2008). Goetz and colleagues, reported that Cav1 expression in fibroblasts remodeled the extracellular matrix to regulate cell shape and to stimulate migration and invasion of cancer cells in vitro, and promote tumor growth and metastasis in vivo in a p190RhoGAP dependent manner (Goetz et al., 2011).

In order to specifically assess the role of Cav1 in the tumor stroma on intratumoral microenvironment remodeling and metastasis, Goetz and colleagues utilized a subcutaneous xenograft model in which primary mouse embryonic fibroblasts (pMEFs) derived from either wild type (WT) or Cav1 knock out animals (Cav1 KO) were coinjected subcutaneously with luminescent LM-4175 metastatic breast tumor cells (Minn et al., 2005) with Matrigel into nude mice (Goetz et al., 2011). Figure 7Ca presents a graphical illustration of the experimental procedure. Primary tumor growth and metastatic tumor growth was assessed by bioluminescent imaging in vivo or ex vivo in extracted organs. Further, immunostaining of primary tumors was utilized to analyze the fiber alignment and organization of the tumor stroma in the presence of WT or Cav1 KO pMEFs, as alignment of collagen fibers in the tumor microenvironment has been shown to enhance tumor cell migration and invasion (Amatangelo et al., 2005; Provenzano et al., 2006). It is important to note that the authors also performed shRNA knock down of p190RhoGAP in Cav1 KO pMEFs to show that the effects of Cav1 KO on metastasis could be reversed by knock down of p190RhoGAP in Cav1 KO pMEFs. This replication study will only address the effects of Cav1 expression in tumor stroma and remodeling of the tumor microenvironment matrix, and will not replicate the effects of p190RhoGAP in this model.

These experiments utilize isolated WT or Cav1 KO pMEFs, which are generated in Protocol 1. The loss of Cav1 in the Cav1 KO pMEFs (Figure 7Ca), lead to an increase in smooth muscle actin (SMA) compared to Cav1 WT pMEFs, which is an indicator of increased activation and extra cellular matrix (ECM) remodeling capabilities of the generated pMEFs (Supplemental Figure S2A) (Goetz et al., 2011). These pMEFs were used in a subcutaneous tumorigenicity assay, with representative bioluminescent images of primary tumors in vivo or ex vivo in extracted organs reported in Figure 7Cc. Bioluminescence of the primary tumors or metastatic foci in each organ was quantified and shown in Figure 7Cb or Supplemental Figure S7Ca. Goetz and colleagues evaluated metastatic tumor growth where they reported an increase in metastatic foci when tumor cells were coinjected with WT pMEFs compared to tumor cells alone or tumor cells coinjected with Cav1 KO pMEFs (Goetz et al., 2011). While not the main focus of this experiment they did not observe a difference in primary tumor growth between these conditions (Goetz et al., 2011). This experiment is important to replicate because it tests the central tenet of the paper, namely that Cav1 expression in stromal fibroblasts contributes to increased metastasis of tumors. This experiment is replicated in Protocol 3.

Although there was no observed difference in primary tumor growth when Cav1 was absent in the tumoral stroma specifically, a decrease in primary tumor growth was observed when the whole mammary gland was deficient for Cav1 in mammary gland allografts and xenografts (Goetz et al., 2011). In a related study, subcutaneous injection of B16 melanoma cells in Cav1 KO mice resulted in a reduced tumor growth compared to injection of tumor cells in WT mice (Chang et al., 2009). Another study, which focused on the size of the primary tumors opposed to metastasis, reported that intradermal coinjection of nude mice with B16F10 melanoma cells and Cav1 KO neonatal dermal fibroblasts increased primary tumor growth when compared to coinjection of tumor cells with WT fibroblasts (Capozza et al., 2012). While no known direct replications of the original study have been reported, several studies have assessed the role of stromal Cav1 expression in different types of tumors, with some studies reporting high Cav1 expression correlated with poor patient survival (Linke et al., 2010; Goetz et al., 2011; Righi et al., 2014), and others reporting low Cav1 expression in the stroma negatively correlated with survival (Simpkins et al., 2012; Ma et al., 2013; Zhao et al., 2013; Ren et al., 2014).

Figure 7Cc and Supplemental Figure S7Cb show representative images of tumor sections immunostained for fibronectin, the CAF marker SMA, and nuclei. Goetz and colleagues showed that fibronectin fiber alignment increased in the presence of Cav1 (Goetz et al., 2011). Supplemental Figure S7Cc shows the results of quantification of fibronectin and SMA fiber alignment as well as cell shape. Goetz and colleagues also show in Figure 7Cd that metastasis correlates with the degree of fibronectin fiber alignment in the tumor stroma. These figures are important to replicate as they show that Cav1 expression in fibroblasts contributes to remodeling of the tumor microenvironment in vivo, and that this remodeling correlates with the amount of metastasis. These experiments are replicated in Protocol 4.

Materials and methods

Protocol 1: isolation of Cav1 WT and Cav1 KO primary MEFs

This experiment describes the isolation of primary MEFs (pMEFs) that will subsequently be used in Protocols 2 and 3.

Sampling

■ Each experiment has 2 cohorts:

○ Cohort 1: Cav1-WT embryos.

○ Cohort 2: Cav1-KO embryos.

■ Experiment performed with three pregnant females in each cohort to ensure enough pMEFs are obtained.

○ Power calculations are not applicable.

Materials and reagents

Procedure

Notes

This protocol contains information described in Cerezo et al. (2009), Razani et al. (2001), and Kamijo et al. (1997).

pMEFs grown in complete DMEM: DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere at 5% CO2.

Sacrifice E14.5 pregnant Cav1WT (B6129SF2/J) and Cav1KO (CAV1/J) mouse by cervical dislocation.

Dissect uterine horns and transfer to tube with ice-cold PBS to a tissue culture hood.

Transfer uterine horns to a 100 mm tissue culture dish; separate embryos from and discard placenta and embryonic sac.

For each embryo, place in a dry 100 mm plate, dissect and discard head and red organs.

Mince with sterile scissors or a sterile razor blade until homogeneous and pipettable.

Add 1 ml 0.05% trypsin/0.53 mM EDTA per embryo, disaggregate further by pipetting, and incubate 5 min at 37°C.

Plate 1 ml of cells/trypsin into 150 mm tissue culture dishes (one dish per embryo), and try to further disaggregate by pipetting.

a. Ensure medium is pre-warmed before adding cells.

b. These are ‘passage 1’ cells.

Repeat the disaggregation with the pipette every 10 min, 3 or 4 times (steps 6–7).

Change the medium after 24 hr.

On day 3 and every third day thereafter, passage 3 × 105 cells per 60 mm dish.

a. Use pMEFs before passage 5.

Use pMEFs in further experiments below.

a. Western blot analysis of Cav1 and SMA levels (Protocol 2).

b. Subcutaneous tumorigenicity assay (Protocol 3).

Repeat for each pregnant mouse.

Deliverables

■ Data to be collected:

○ Mouse health records (gender of mice, age of embryos when sacrificed).

■ Sample delivered for further analysis:

○ pMEFs derived from Cav1WT and Cav1KO mouse embryos (Protocols 2 & 3).

Confirmatory analysis plan

This protocol will not perform any statistical tests.

Known differences from the original study

All known differences, if any, are listed in the materials and reagents section above with the originally used item listed in the comments section. The comments section also lists if the source of original item was not specified. All differences have the same capabilities as the original and are not expected to alter the experimental design.

Provisions for quality control

All of the raw data, will be uploaded to the project page on the OSF (https://osf.io/7yqmp) and made publically available.

Protocol 2: assessment of Cav1 and SMA levels in pMEFs by western blot

This experiment assesses the protein levels of Cav1 to ensure the Cav1 KO pMEFs generated are actually knockout for Cav1. Additionally, SMA is assessed to determine if levels in Cav1 KO pMEFs are increased over Cav1 WT pMEFs, which indicate increased activation and ECM remodeling capabilities of the generated pMEFs. It is similar to the experiments reported in Figure 7Ca and Supplemental Figure S2A.

Each experiment has two cohorts:

○ Cohort 1: Cav1-WT pMEFs.

○ Cohort 2: Cav1-KO pMEFs.

Each clone of pMEFs will be assessed for the following markers:

○ Cav1.

○ SMA.

○ γ-tubulin.

Experiment will be conducted once and used to assess which clones will be utilized.

○ Power calculations are not applicable.

Note

pMEFs are generated in Protocol 1.

pMEFs maintained in: DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere at 5% CO2.

Prepare each clone of pMEFs for lysis.

a. Wash cells with ice-cold PBS and remove excess PBS.

b. Add cold RIPA buffer to cells (use 1 ml of buffer per 60 mm dish).

c. Keep on ice for 5 min, swirling plate occasionally for uniform spreading.

d. Collect lysate with cell scraper and transfer to microcentrifuge tube.

e. Centrifuge samples at ∼14,000×g for 15 min at 4°C.

f. Transfer supernatant to new tube.

Determine protein concentration using Bradford assay following manufacturer's instructions and a BSA standard curve.

Adjust protein concentration and prepare up to 70 µg/lane of total cell lysate by adding 6× SDS-PAGE sample buffer and heating to 100°C for 5 min.

Separate samples and molecular weight marker by SDS-PAGE gel electrophoresis in 1× tris-glycine SDS buffer following replicating lab's protocol. Run at 100V through the stacking part of the gel and up to 200 V after the proteins have migrated through the resolving gel. Allow migration to continue until the blue dye front is at the bottom of the gel, but has not migrated off.

a. Include Cav1 WT and Cav1 KO clones on same gel to allow comparison.

Transfer gel to a PVDF membrane, following replicating lab's transfer procedure.

After the transfer, stain the membrane with Ponceau S to visualize the transferred protein. Image membrane, than destain in ddH2O and rinse with TBS buffer.

Incubate membrane with 5% non-fat dry milk in TBST buffer.

a. TBST buffer: TBS with 0.1% Tween-20.

Probe membrane with the following primary antibodies diluted in 5% non-fat dry milk in TBST buffer:

a. mouse anti-Cav1; use at 1:1000; 21 kDa.

b. mouse anti-SMA; use at 1:1000; 42 kDa.

c. mouse anti-γ-tubulin; use at 1:1000; 48 kDa.

Wash membrane in TBST buffer.

Detect primary antibodies with the following secondary antibody diluted in 5% non-fat dry milk in TBST buffer:

a. anti-mouse-HRP; use at 1:5000 to 1:10,000.

Wash membrane in TBST buffer.

Detect signal with ECL reagent following manufacturer's instructions.

Image the entire membrane including molecular weight ladder.

Quantify signal intensity.

a. For each antibody subtract background intensity from values and then divide by the γ-tubulin loading control.

b. Calculate the normalized SMA levels for all clones.

c. Confirm absence of Cav1 protein in all Cav1 KO pMEFs.

Exclude any clones that do not display a presence (Cav1 WT pMEFs) or absence (Cav1 KO pMEFs) of Cav1. Exclude any clones that do not have an increase in SMA expression with a loss of Cav1 (Cav1 KO pMEFs compared to Cav1 WT pMEFs).

Use remaining pMEF clones in further experiments before passage 5:

a. Subcutaneous tumorigenicity assay (Protocol 3).

Deliverables

■ Data to be collected:

○ Images of probed membranes (full images with ladder).

○ Raw and quantifed signal intensities normalized for γ-tubulin loading and total protein levels.

■ Sample delivered for further analysis:

○ Cav1 WT and Cav1 KO pMEFs that are included for further use (Protocol 3).

This protocol will not perform any statistical tests.

Known differences from original study

The replicating lab western blot protocol will be used. All known differences, if any, are listed in the materials and reagents section above with the originally used item listed in the comments section. The comments section also lists if the source of original item was not specified. All differences have the same capabilities as the original and are not expected to alter the experimental design.

Transfer quality will be assured by Ponceau staining. All of the raw data, will be uploaded to the project page on the OSF (https://osf.io/7yqmp) and made publically available. This experiment is also the quality control for the pMEFs generated in Protocol 1 that will be utilized in Protocol 3 to assess Cav1 status and ECM remodeling capabilities.

Protocol 3: subcutaneous tumorigenicity assay of tumor cells co-injected into athymic nude mice with pMEFs

This experiment tests the contribution of Cav1 expression in pMEFs on tumorigenicity and metastasis of breast cancer cells. pMEFs derived from WT or Cav1 KO animals are co-injected with LM-4175 breast cancer cells in a Matrigel plug subcutaneously in nude mice and tumor growth and metastasis are monitored by bioluminescent imaging. It is a replication of the experiment reported in Figure 7Cb and Supplemental Figure S7Ca.

■ Experiment has 3 cohorts:

○ Cohort 1: LM-4175 cells alone.

○ Cohort 2: LM-4175 cells co-injected with Cav1 WT pMEFs.

○ Cohort 3: LM-4175 cells co-injected with Cav1 KO pMEFs.

■ Experiment will analyze the following number of mice per cohort for a minimum power of 80%:

○ See Power calculations section for details.

Cohort 1: 7 mice.

Cohort 2: 21 mice.

Cohort 3: 21 mice.

■ To account for unexpected euthanasia of mice before the end of the experiment, 20% more mice were added to ensure the needed number of mice survive each cohort:

○ Cohort 1: 9 mice.

○ Cohort 2: 26 mice.

○ Cohort 3: 26 mice.

pMEFs are generated in Protocol 1 with inclusion/exclusion criteria determined in Protocol 2.

pMEFs maintained in: DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere at 5% CO2.

LM-4175 tumor cells express HSV-tk1-GFP-Fluc and will be sent for mycoplasma testing and STR profiling as well as screened against a Rodent Pathogen Panel.

LM-4175 maintained in: normal glucose DMEM supplemented with 10% FBS at 37°C in a humidified atmosphere at 5% CO2.

Athymic nude mice should be 8–10 weeks old when they arrive and about 10–12 weeks old when injected with cells.

Cell preparation for injection:

a. LM-4175 tumor cells alone:

i. Prepare 1 × 107 cells/ml of LM-4175 tumor cells in sterile chilled PBS.

You will need 0.1 ml (1 × 106 cells) per injection.

Adjust cell mixture accordingly for needed number of injections.

ii. Mix cell suspension.

b. LM-4175 tumor cells with pMEFs.

i. Prepare 2 × 107 cells/ml of LM-4175 tumor cells in sterile chilled PBS.

ii. Prepare 2 × 107 cells/ml of Cav1 WT pMEFs, or Cav1 KO pMEFs in sterile chilled PBS.

iii. Mix the two suspensions together to form a 1:1 mixture.

You will need 0.1 ml (2 × 106 cells) per injection.

Adjust cell mixture accordingly for needed number of injections.

c. Using well-chilled tubes and pipette tips, mix each final cell suspension with an equal amount (0.1 ml) of Matrigel and store on ice.

Anesthetize 10–12 week old female athymic nude mice.

a. House mice under pathogen-free conditions and give autoclaved food and water ad libitum.

b. Anesthetize with 100 mg/kg ketamine and 10 mg/kg xylazine.

Using a 25 G needle and 1 ml syringe, aspirate 0.2 ml of Matrigel/cell suspension and inject subcutaneously at the prepared site.

a. During injection keep the mice warm by lying them on a warming plate designed for animal experiments.

b. Stretch the abdominal cavity avoiding the formation of wrinkles.

c. Inject Matrigel/cell suspension slowly by introducing the needle as parallel as possible to the skin. Ensure the Matrigel/cell suspension forms a visible bump under the skin.

d. If injection enters the peritoneum, there will not be a bubble in the skin and instead of formation of one primary tumor there will be many intraperitoneal tumors. These mice should be excluded from the experiment and analysis.

i. It is important not to take the needle out of the skin until the Matrigel has gelled. Gelification takes a very short time at body temperature (∼1 min).

Allow mice to recover and keep them warm until they wake up.

Maintain mice for 70 days.

a. Measure tumor size twice weekly with precision calipers.

b. Initial tumor formation is defined as the time when the tumor reaches a diameter of 3 mm. The tumor should never be greater than 10% body weight or exceed 20 mm in any one dimension. If ulceration or infection at the tumor site, or interference with eating or impairment of ambulation by the tumor occurs, the animals should be euthanized.

c. Additional euthanasia criteria to ensure no animal suffering (when two or more of these criteria are detected, monitor the animals for 12 hr, if the condition does not improve, euthanize):

i. Rapid or progressive weight loss (more than 10% in 2 weeks).

ii. Debilitating diarrhea.

iii. Dehydration/reduced skin turgor.

iv. Edema.

v. Sizable abdominal enlargement or ascites.

vi. Hunched posture.

vii. Lethargy.

viii. Labored breathing, nasal discharge.

ix. Bleeding from any orifice.

x. Any condition interfering with daily activities for more than 2 hr (e.g., eating or drinking, ambulation, or elimination).

xi. Excessive or prolonged hyperthermia or hypothermia.

At 70 days (or an earlier time point if the number of mice euthanized in step 5 compromise the ability to obtain enough mice for analysis), anesthetize each mouse and inject pairs of mice with 150 µl of 17.5 mg/ml luciferin solution intraperitoneally.

a. Anesthetize with 100 mg/kg ketamine and 10 mg/kg xylazine.

b. Prepare solution of luciferin according to manufacturer's instructions.

c. Within each pairing of mice image, euthanize, dissect, re-image, and freeze tumors (steps 6–10) from mice from different cohorts in parallel (i.e., one from each of the three cohorts, or one each from cohort 2 and cohort 3) so variations during the procedure are equal across cohorts.

After 20 min post luciferin injection, place in IVIS Imaging System and take ventral views for photon flux quantification.

a. To facilitate metastasis detection in axillary/brachial lymph nodes, secure front limbs with tape, and shield the lower portion of the animal to block bioluminescence from the primary tumors.

b. Use extended exposures (0.2 s–20 s) for in vivo metastases detection.

c. To detect well-defined in vivo metastases, shield lower portion of the animal to block bioluminescence from the primary tumors and bladder, and use a 20 s exposure.

Euthanize mouse and excise the primary tumor and extract organs.

a. Before euthanasia, inject a second dose of luciferin solution—50 µl of 17.5 mg/ml luciferin solution intraperitoneally.

b. Wait 20 min, euthanize mouse and quickly excise the primary tumor and extract the following organs to image for metastasis:

i. Lymph nodes.

ii. Spleen.

iii. Lungs.

iv. Liver.

v. Intestines.

vi. Kidneys.

Reimage organs ex vivo in IVIS imaging system.

a. Place all organs in plastic dish (uncovered), with organs separated, for imaging.

b. Reimage organs as quickly as possible after organ dissection.

c. Acquire multiple exposure times to manually quantify every visible metastatic focus. (Recommend 1, 20, and 60 s exposures, with 2 min as longest exposure time).

d. Small metastatic foci can be detected by adjusting the scale of photon flux in Living Image software (version 3.2).

Cut extracted primary tumors in half and freeze in O.C.T. compound for further analysis (Protocol 4). Randomly select the following number of tumors from each cohort:

a. LM-4175 cells alone—4 tumors.

b. LM-4175 cells co-injected with Cav1 WT pMEFs—7 tumors.

c. LM-4175 cells co-injected with Cav1 KO pMEFs—7 tumors.

■ Data to be collected:

○ Passage of cells, particularly pMEFs, injected in each mouse.

○ Mouse health records (including if euthanasia is required and reason, date of euthanasia, and date of imaging if it is not 70 days).

○ Twice weekly tumor measurements.

○ All images of mice in vivo to detect primary tumor (compare to Figure 7Cc).

○ All images of excised organs (ex vivo) to detect metastatic foci (compare to Figure 7Cc).

○ Raw photon flux measurements of primary tumor and metastasis in vivo and metastatic foci ex vivo, including counts of metastatic foci from ex vivo images.

○ Graph of primary tumor growth (photon flux (P/s, ×1010) for all conditions (compare to Figure S7Ca).

○ Graph of metastatic foci (ex vivo bioluminescence) per mouse for all conditions. One graph for each organ type and for total organs (compare to Figure 7Cb).

■ Sample delivered for further analysis:

○ Preserved primary tumors for further analysis in Protocol 4.

This replication attempt will perform the following statistical analysis listed below:

■ Statistical analysis:

○ Wilcoxon–Mann Whitney test of total metastatic foci counts per mouse by ex vivo bioluminescence for the following comparisons with the Bonferroni correction:

Note: in order to enable a direct comparison to how the original data was analyzed, uncorrected tests will also be performed.

LM-4175 only to LM-4175 co-injected with Cav1 WT.

LM-4175 only to LM-4175 co-injected with Cav1 KO.

LM-4175 co-injected with Cav1 WT to LM-4175 co-injected with Cav1 KO.

○ Kruskal–Wallis test of primary tumor growth per mouse by in vivo bioluminescence for all conditions.

■ Meta-analysis of effect sizes:

○ Compute the effect sizes of each comparison, compare them against the reported effect size in the original paper and use a random effects meta-analytic approach to combine the original and replication effects, which will be presented as a forest plot.

The replication attempt is not including the experimental condition of p190RhoGAP knockdown in Cav1 KO pMEFs. All known differences, if any, are listed in the materials and reagents section above with the originally used item listed in the comments section. The comments section also lists if the source of original item was not specified. All differences have the same capabilities as the original and are not expected to alter the experimental design.

The pMEFs used in this protocol were assessed for Cav1 status and ECM remodeling capabilities (SMA levels) in Protocol 2. The cell lines used in this experiment will undergo STR profiling to confirm their identity and will be sent for mycoplasma testing to ensure there is no contamination. Additionally, cells will be screened against a Rodent Pathogen Panel to ensure no contamination prior to injection. Mice will be injected with luciferin, imaged, euthanized, dissected, re-imaged, and tumors frozen one animal from each cohort done in parallel to have variations during the procedure equal across cohorts. All of the raw data, including the image files and quantified metastatic foci, will be uploaded to the project page on the OSF (https://osf.io/7yqmp) and made publically available.

Protocol 4: examining intratumoral fiber orientation and cell shape in tumor cells co-injected into nude mice with pMEFs

This experiment tests the effect of Cav1 expression in pMEFs on fiber orientation and cell shape within the tumor environment. Tumor sections derived from Protocol 3 are immunostained for fibronectin and smooth muscle actin, and the orientation of the fibers and cell shape are quantified. Fibronectin fiber orientation is further correlated with the amount of metastasis. It is a replication of experiments reported in Figure 7Cd and Supplemental Figure S7Cc.

■ Experiment has 3 cohorts:

○ Cohort 1: LM-4175 cells alone.

○ Cohort 2: LM-4175 cells co-injected with Cav1 WT pMEFs.

○ Cohort 3: LM-4175 cells co-injected with Cav1 KO pMEFs.

■ Stained for:

○ Fibronectin.

○ SMA.

○ Control staining:

Isotype control.

Secondary antibody only control.

■ Experiment will use the following number of primary tumors with 2 sections stained per tumor, with additional sections for controls, and 5 regions imaged per section for a minimum power of 80%:

○ See power calculations section for details.

Cohort 1: 4 tumors.

Cohort 2: 7 tumors.

Cohort 2: 7 tumors.

■ Experiment will analyze at least the following number of SMA + cells from the following cohorts for a minimum power of 80%:

○ The original lab collected an average of 10–20 cells/region.

○ The replication will collect 5 regions/tumor.

○ See Power calculations section for details.

Cohort 1: 85 images.

Cohort 2: 85 images.

Cohort 3: 65 images.

Cut primary tumor from Protocol 3 on a microtome with a section thickness of 8 µm and mount on slides.

a. Cut at least 2 sections from each tumor.

Fix and permeabilize sections:

a. Place sections in −20°C acetone and incubate at −20°C for 5 min.

b. Place sections in −20°C acetone:chloroform (1:1) solution and incubate at −20°C for 5 min.

c. Place sections in −20°C acetone and incubate at −20°C for 5 min.

d. Wash sections 2 times in 1× PBS.

Incubate sections in 1× PBS supplemented with 2% BSA for 20 min at room temperature. Wash 2 times in 1× PBS.

Incubate sections in both primary antibodies diluted in 1× PBS supplemented with 2% BSA overnight at 4°C. Include additional sections for controls.

a. Rabbit-anti-fibronectin; use at 1:200 dilution.

b. Mouse-anti-αSMA; use at 1:100 dilution.

c. Control staining conditions:

i. Isotype control; use at same concentration as primary.

ii. secondary antibody only controls.

Wash 2 times in 1× PBS.

Incubate sections in secondary antibody and Hoechst dye diluted in 1× PBS supplemented with 2% BSA for 1 hr at 37°C.

a. Alexa 594 donkey-anti-rabbit; use at manufacturer's recommended dilution.

b. Alexa 647 donkey-anti-mouse; use at manufacturer's recommended dilution.

c. Hoechst dye; use at 1:1000 dilution.

Randomly image 5 independent regions per section at 20× magnification using a confocal microscope.

Note: if sections are unable to be imaged due to necrosis, autofluorescence, low fibronectin deposition, or damage during the staining procedure, take images, and exclude from analysis with indicated reason.

a. Total number of regions per tumor is 5 (not including control images).

b. Acquire z-stack for each region with 0.5 µm-thick z-slices.

c. Use a 20× objective with a minimum numerical aperture (NA) of 0.70.

Blindly quantify intratumoral orientation of fibronectin fibers and SMA + cells and elliptical form factor of SMA + cells. For details see Amatangelo and colleagues (Amatangelo et al., 2005).

Note: if images are unable to be evaluated due to necrosis, autofluorescence, low fibronectin deposition, or damage during the staining procedure, record as such.

a. Overlay z-slices to make reconstituted views of the corresponding 3-D fibers for each region.

b. Subject reconstituted 3-D projections to identical modification and digital filters as described below using MetaMorph offline 6.2r1 imaging analysis software.

c. Reduce non-specific background with the Flatten Background function by selectively darkening objects with a pixel area greater than 15.

d. Create a binary image by selecting a 35% threshold at the maximum internal intensity option using the internal threshold function.

e. Count all fibers recognized, as well as their orientation (angle relative to x-axis) using the integrated morphometry analysis function. Use auto-threshold for light objects and measure angle of displayed objects.

f. Approximate relative angles to the nearest 10th degree using the rounding function on Microsoft's Excel software and determine the mode angle for each image. Arbitrarily set the mode angle to 0° for each image.

g. Quantify the elliptical form factor (EF = length/breadth) of SMA + cells using the integrated morphometry analysis function.

■ Data to be collected:

○ Image stacks of fibronectin, SMA, and Hoescht-stained tumor sections for all conditions, including controls and any images taken, but unable to be analyzed (compare to Figure 7Cc and S7Cb).

○ Raw data and summary data of fibronectin fiber orientation (compare to Figure S7Cc).

○ Raw data and summary data of elliptical form factor for SMA + cells (compare to Figure S7Cc).

○ Raw data and summary data of SMA + cells fiber orientation (compare to Figure S7Cc).

This replication attempt will perform the following statistical analysis listed below:

■ Statistical analysis:

○ Wilcoxon-Mann Whitney test of percent of fibronectin fibers within ± 20° per tumor by for the following comparisons with the Bonferroni correction:

Note: in order to enable a direct comparison to how the original data was analyzed, uncorrected tests will also be performed.

LM-4175 only to LM-4175 co-injected with Cav1 WT.

LM-4175 co-injected with Cav1 WT to LM-4175 co-injected with Cav1 KO.

○ Wilcoxon–Mann Whitney test of elliptical form factor per SMA + cells for the following comparisons with the Bonferroni correction:

LM-4175 only to LM-4175 co-injected with Cav1 WT.

LM-4175 co-injected with Cav1 WT to LM-4175 co-injected with Cav1 KO.

○ Wilcoxon–Mann Whitney test of percent of SMA + cells fibers within ± 20° per tumor by for the following comparisons with the Bonferroni correction:

LM-4175 only to LM-4175 co-injected with Cav1 WT.

LM-4175 co-injected with Cav1 WT to LM-4175 co-injected with Cav1 KO.

○ Spearman's rho correlation of intratumoral fibronectin fibers within ± 20° vs number of total metastasis per mouse (cohorts combined).

■ Meta-analysis of effect sizes:

○ Compute the effect sizes of each comparison, compare them against the reported effect size in the original paper and use a random effects meta-analytic approach to combine the original and replication effects, which will be presented as a forest plot.

The replication attempt is not including the experimental condition of p190RhoGAP knockdown in Cav1 KO pMEFs. All known differences, if any, are listed in the materials and reagents section above, indicated by an asterisk, with the originally used item listed in the comments section. The comments section also lists if the source of original item was not specified. All differences have the same capabilities as the original and are not expected to alter the experimental design.

Isotype and secondary antibody only controls will be included. If a section or image is unable to be quantified, due to necrotic damage, autofluroescence, low fibronectin deposition, or damage during the staining procedure, this data will be excluded from the analysis, similar to the original study, but recorded. The objective used to acquire the original images, that were subsequently used for the analysis was a Leica HCX PL APO CS 20×/0.70 IMM UV (communication with original authors). The replication will use a 20× objective with a minimum NA of 0.70. Images will be blindly and randomly taken and evaluated, and all of the raw data, including the control images and analysis files, will be uploaded to the project page on the OSF (https://osf.io/7yqmp) and made publically available.

Power calculations

For additional details on power calculations, please see analysis scripts and associated files on the Open Science Framework:■ https://osf.io/q3e4u/.

Protocols 1 and 2

Not applicable.

Protocol 3

Total metastatic foci per mouse

Summary of original data (provided by original authors).

Power calculations performed with G*Power software, version 3.1.7. (Faul et al., 2007).

Test family

■ t test: means: Wilcoxon–Mann–Whitney test (two groups, one tail), alpha error = 0.0167.

Primary tumor growth

Summary of original data (provided by original authors).

Analysis of original data: (Kruskal–Wallis; performed with GraphPad Prism, version 6.0).

Power calculations performed with G*Power software, version 3.1.7. (Faul et al., 2007).

■ F test: ANOVA: Fixed effects, omnibus, one-way, alpha error = 0.05.

Protocol 4

Percent of fibronectin fibers within ± 20°

Summary of original data (obtained from Figure S7Cc).

Power calculations performed with G*Power software, version 3.1.7. (Faul et al., 2007).

■ t test: Means: Wilcoxon–Mann–Whitney test (two groups, one tail), alpha error = 0.025.

Elliptical form factor of SMA + cells

Summary of original data (obtained from Figure S7Cc).

Power calculations performed with G*Power software, version 3.1.7. (Faul et al., 2007).

■ t test: Means: Wilcoxon–Mann–Whitney test (two groups, one tail), alpha error = 0.025.

Percent of SMA + fibers within ± 20°

Summary of original data (obtained from Figure S7Cc).

Power calculations performed with G*Power software, version 3.1.7. (Faul et al., 2007).

■ t test: means: Wilcoxon–Mann–Whitney test (two groups, one tail), alpha error = 0.025.

Correlation of percent of fibronectin fibers within ± 20° and number of metastasis

Original data (obtained from original authors).

The shared data contained 27 XY pairs with a calculated Spearman rho of 0.7488, which is missing 3 pairs included in the published analysis, with a Spearman rho of 0.81. The power calculations were performed on the shared data and Spearman rho, which will give a more conservative sample size to detect the published value.

Power calculations performed with R software, version 3.1.0 (R Development Core Team, 2014).

■ Correlation: Spearman's rho test (one sided), alpha error = 0.05.

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Thank you for sending your work entitled “Registered report: Biomechanical remodeling of the microenvironment by stromal Cav1 favors tumor invasion and metastasis” for consideration at eLife. Your article has been evaluated by Fiona Watt (Senior editor), a Reviewing editor, and four reviewers, one of whom has direct statistical expertise.

The following individuals responsible for the peer review of your submission have agreed to reveal their identity: Miguel Del Pozo (Reviewer 1), Peter Friedl (Reviewer 2), and Dawn Teare (Reviewer 4). Reviewer 3 remains anonymous.

The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.

Overall, the reviewers agree on the choice of the key experiments and the experimental approach. However, a number of points have been raised that need to be addressed in a revised Registered Report before proceeding with the experiments:

The authors should provide a more extensive and more balanced discussion of the literature on the function of caveolin-1 in different cancers; some missed references are listed in the comments of reviewer 1 below. Importantly, the Capozza et al. paper, presented as in contradiction with the findings by Goetz et al., may not be in contradiction since Capozza et al. focused on the size of the primary tumours whereas Goetz et al. focused on metastasis (see also comments from Reviewer 1 below for more details). This should be clarified. If the authors wish to insist on a discrepancy between the Capozza et al. and the Goetz et al. studies, it would be a good idea to inject the B16F10 cells used by Capozza et al. (Reviewer 3's suggestion), to clarify whether the discrepancy may be caused by a difference in cell lines.

There are a number of experimental points listed in the reviews comments appended below that need to be clarified.

Extracts from Reviewer #1:

A significant overstatement made by the authors of this Reproducibility Project pertains to protocol 2. They claim that there is a contradiction between the tested paper (Goetz et al. Cell 2011) and another one by the Lisanti group: “In contrast to these findings, Capozzo and colleagues showed that intradermal coinjection of nude mice with B16F10 melanoma cells and Cav1 KO neonatal dermal fibroblasts increased primary tumor growth when compared to coinjection of tumor cells with WT fibroblasts (Capozza et al., 2012).” We disagree that there is conflict here, since the original paper by Goetz et al. was focused on studying changes in stromal remodeling and, as a consequence of them, correlative changes in tumor local invasion and distant metastasis. The growth of the primary tumor was not the main focus of Goetz et al., and hence it was not as critically evaluated as ECM remodeling or metastasis, as it was the case for Capozza et al. Due to the nature of the allograft/xenograft experiments of Figure 7, focused on evaluating ECM remodeling and the appearance of metastasis, it was also possible to measure the size of the primary tumor, but this was not as critically monitored as the metastasis formation. Hence, we believe that it is not correct to claim that one of the main conclusions of the original paper is that the growth of the primary tumor is not affected by the absence of Cav1 expression in fibroblasts. Therefore, we believe is not correct to include primary tumor growth as one of the statistical analysis to be performed in protocol 2. In the experimental approach that Fiering et al. have chosen, the primary tumor growth was not significantly different between any of the studied conditions. However, primary tumor growth was decreased in the absence of Cav1 expression in mammary gland allografts (Figure 7A and S7A) and in mammary gland xenografts (Figure 7B and S7B). It is important to note that in these approaches, the whole mammary gland was deficient for Cav1 expression, not only the fibroblasts, and hence they are not exactly comparable. In the same regard, Capozza et al. used neonatal dermal fibroblasts, which are not exactly the same as MEFs. Further, Fiering et al. fail to cite other papers that apparently contradict Capozza et al. and are in line with results of Figure S7A and S7B. One example comes from the Dvorak group, which showed that subcutaneous injection of B16 melanoma cells in Cav1 KO mice leads to reduced tumor growth compared to injection of tumor cells in WT mice (Chang et al., Am J Path 2009). Fiering et al. should also cite this work, but, even more importantly, these differences highlight the complexity of this biological problem, and the potential difficulty to reproduce observations made in several labs.

In the particular case of the subcutaneous xenograft model (Figure 7C), the main conclusions to be reproduced should thus be:

1) Absence of Cav1 expression in fibroblasts inhibits metastasis of LM-4175 breast tumor cells.

2) Absence of Cav1 expression in fibroblasts results in a less anisotropic ECM (with a lower proportion of parallel Fibronectin fibers).

3) A statistical significant correlation between the anisotropy of the intratumoral fibers and the total number of metastasis per mouse (Spearman's rho correlation).

4) Presence or absence of Cav1 expression in fibroblast modifies cell shape (elliptical factor of SMA+ cells).

Do the authors accurately summarize the literature, especially with respect to other direct replications? No, and this is a critical issue that we have observed in reviewing the manuscript by Fiering et al. We do not agree with the selection of papers made to introduce the subject of Caveolin-1 expression in tumor stroma. The papers chosen belong mostly to the same group, which consistently reports Caveolin-1 as a tumor suppressor. However, caveolin-1 expression in cancer is a very controversial subject of research. There are thousands of papers and one can virtually find papers showing both increased and decreased expression in any particular type of cancer (Parton and del Pozo, Nature Reviews Mol Cell Biol 2013). A review that carefully evaluated caveolin-1 expression in multiple types of cancers from primary to metastatic stages, concluded a general trend in which Caveolin-1 appears to act as a tumor suppressor at early stages of cancer progression, but it is up-regulated in several multidrug-resistant and metastatic cancer cell lines and human tumor specimens, positively correlating with tumor stage and grade in numerous cancer types (Shatz and Liscovitch, Int J Radiat Biol 2008). One of the conclusions of the original article by Goetz et al. was that part of this variability could stem from Cav1 expression in stromal rather than tumor cells. In fact, the papers cited in the introduction plus some others are already confirming this.

Therefore, a more thorough review of the existing literature should be performed in order to better present the complexity of this particular aspect. For example, loss of Cav1 function in stromal cells of various organs leads to benign stromal lesions responsible for abnormal growth and differentiation of the epithelium and to dramatic reductions in life span (Yang et al., Exp Mol Path 2008). Consistently, Goetz et al. showed that Cav1 expressed in fibroblasts can modulate normal cell morphology via force-dependent ECM remodelling. Thus, reports like Yang et al. showing that lack of stromal Cav1 could disturb normal tissue architecture should also be cited by Fiering et al.

Another important point is that, in general, the papers cited in the Introduction do not use specific markers of specific stromal types, so it is not possible to be certain whether they are CAFs or some other stromal cell type. Goetz et al. used several fibroblast markers throughout the study, and in particular Cav-1/α-SMA colocalization in the human cancer samples. These differences should be outlined by Fiering et al.

To the best of our knowledge, no direct replications of the original study have been reported. However, several recent studies (not included by the authors) have addressed the role of stromal Cav1 expression in different types of tumors. Righi et al. have recently published that high Cav1-expression in the stroma is associated with a worse patient outcome in malignant pleural mesothelioma (Righi et al., Am J Clin Pathol 2014), although the molecular mechanism behind this conclusion was not experimentally addressed. These results are in line with the data of the original article on a breast cancer tissue microarray analysis (Figure 5C) showing that increased Cav1 in the stroma correlated with decreased survival. Moreover, Linke et al. have reported that stromal Cav1 (often surrounding “nests” of tumor cells) was a powerful univariate prognostic marker that remained significant in the context of a pre-existing multi-marker Breast Cancer diagnostic commercial profile (Linke SP, Krajewski S, Bremer TM, Man AK, Zeps N, Spalding L. “Stromal caveolin-1 is a powerful marker that further enhances a multi-marker prognostic profile”. Cancer Res. 2010;70(24 Suppl):Abstract P3-10-42). Further, results presented in 2014 ASCO Annual Meeting by the Spanish Breast Cancer Research Group (GEICAM) confirm positive stromal CAV-1 as a prognostic breast cancer marker.

In contrast, another study focused of gastric cancer reported that low stromal-Cav1 expression was associated with worse patient outcome (Zhao et al., PLoS One 2013). The molecular mechanism was not addressed and the differences with previously published results (including the original study) poorly discussed, but this already shows the tremendous variability existing in this complex subject.

As mentioned in the previous section, when referring to the Capozza et al. paper the authors should also cite the work of the contrasting paper by the Dvorak group (Chang et al., Am J Path 2009). As in the other examples mentioned throughout this section, it is not clear why Fiering et al. only cite those papers in apparent contrast with the original article.

Therefore, the authors of this Reproducibility Project should undertake a comprehensive review of the literature on this subject. In the current version they have only chosen those papers that somehow contradict the original one, but they do not mention to what extent they are different (type of tumor, TMA vs. mouse experiments, etc.) and missed important literature that should be included, some of which is consistent with Goetz et al.

Are the proposed experiments appropriately designed? The proposed experiment is appropriately designed, although several small modifications should be highlighted:

1) Protocol 1, procedure points 5-6 do not reflect the original protocol and should be indicated by an asterisk.

2) Protocol 2, it must clearly indicate that the MEFs co-injected are primary MEFs.

3) Protocol 2, procedure point 3b. Nude mice do not require shaving since they do not have any hair; shaving will thus induce unnecessary skin irritation, which could lead to stromal activation.

4) Protocol 2, procedure point 3d. It is important not to take the needle out of the skin till the matrigel has gelified. Gelification takes very short time at body temperature (around a minute).

5) Protocol 2, procedure point 6. Although in the original study the final analysis point was set at 70 days, it is important to keep some kind of flexibility for this end-point. The investigators must consider shortening this time if the number of mice euthanized compromise the success of the whole study (due to the reasons explained in point 5).

6) Protocol 3, sampling. Please stain at least 2 sections per tumor, since some sections are difficult to evaluate due to necrosis, autofluorescence or damage during the staining procedure.

7) Protocol 3, materials. The authors plan to use Alexa488 conjugated donkey anti-rabbit IgG (although later in procedure point 6, they claim to use goat-anti rabbit FITC, please check this inconsistency). In the original study, goat-anti rabbit Cy3 was used. This selection is not trivial, since the plasmid used to express Luciferase in the LM-4175 cells (HSV-tk1-GFP-luc) contains an IRES-GFP sequence. Thus, LM-4175 cells are green (although not really bright), and therefore this fluorescence channel must not be used for additional staining.

8) Protocol 3, materials. The authors plan to use Alexa647 conjugated donkey anti-mouse IgG as we have done. However, later in procedure section, point 6, they claim to use goat-anti mouse CF640R, please check this inconsistency.

Are the proposed statistical analyses rigorous and appropriate? The use of Wilcoxon-Mann-Whitney test to compare total metastatic foci counts in the three groups is well sounded and agrees with the statistical analysis from the original paper; however, since the original paper did report raw p-values only, we believe it would be more appropriate to report also non-corrected p-values in the reviewed paper. On top of that, post-hoc methods based on the control of the FWER (such as Bonferroni) tend to be too conservative compared to methods controlling the FDR, which are the ones we believe should be used by Fiering et al. The use of Bonferroni would significantly dampen the probability to find significant differences, which we believe is somehow unfair for this Reproducibility Project.

On the other hand, Fiering et al. report that they will perform a meta-analysis with the results of the original paper and the new results, but there are no details about the methodology used for such a meta-analysis.

What can the replication team do to maximize the quality of the replication?

Please see the modifications of the protocol highlighted above. Although with 3 regions imaged per sections the authors claim to obtain a minimum power of 80%, some tumors are difficult to image (necrotic damage, autofluorescence, low fibronectin deposition or damage due to preparation or manipulation). For these reasons, we would highly recommend to stain at least two sections per tumor and to image at least five regions per section, although the more the better.

Extracts from Reviewer #2:

Several points not specified in Goetz et al. 2011 should be clarified with the original authors, before initiating the experiments. eLife has verified these points with the original authors and the responses follow the questions:

The order code of the nude mice and their age range when used for the study.

Nude mice, official name from Harlan: Athymic Nude Mouse -Hsd: Athymic Nude-Foxn1nu.

And their age range when used for the study: 8-10 weeks when they arrive, about 10-12 when experiment was done.

Whether DMEM with high glucose was used for in vitro culture of MEFs. Normal DMEM, not high glucose.

Original matrigel stock concentration, so the used relative dilution yields the same final concentration for implantation.

Typical protein concentrations for BD Matrigel Matrix are between 9-12 mg/ml.

Protocol 2:

For metastasis detection, a range of exposure times from 20s to 2 min was used to detect both large and small metastases. Thus, each mouse and excised organ should be assessed to reach maximum sensitivity and count a maximum of micrometastases. To reach high sensitivity, when monitoring the lungs, liver and brain, the lower abdomen (containing tumor and luminescence in the bladder) should be shielded during longer exposure times. Also, for metastasis detection of excised organs, 2 min should be included as longest exposure time.

Protocol 3:

Irrespective of the power calculation, because of expectable intra-tumor variation of stromal density and organization, several sections from the same tumor should be analyzed for fibronectin alignment and cell elongation. The analysis of only 3 fields from a single section may be flawed by accidental sample variability and insufficient representativity.

Extracts from Reviewer #4:

For protocol 2 and 3 the exact details of what was found in the original paper are reported (generally groups means and SDs) so I easily can follow this logic of how the sample size/power calculations have been derived. I have a number of questions regarding assumptions made in these sample size calculations:

1) Often the SD used in the power calc is estimated on a group of size between 6 and 15 so these estimates will tend to be underestimates. This is not so much of a problem when the effect sizes are approximately 1 SD but it could still be taken account of. I cannot replicate the pooled SDs used in the tables – mine come out slightly larger.

2) Sometimes the type 1 error is adjusted for multiple testing and sometimes it is not. If the same animals are studied for several outcomes then these should be treated as multiple testing problems. When several organ sites are examined this must surely be the same animals?

3) It seems odd to me that the replication study wants to replicate results that were not statistically significant in the original study? In these cases they argue that they select an effect size that they have 80% power to detect.

4) There are many hypotheses being tested in this replication. Are they all equally important? Are some of them nested? If half of the tests are statistically significant will this be regarded as a validation of the original report? I am not confident that these sample sizes will have sufficient power for so many repeated tests.

5) There is some confusion in statistical language which may be a feature of the G*power program. The sample size calculations state that t-tests (to detect standardised effects) will be used but the analysis plan uses non-parametric tests (Kruskall Wallis and Mann Whitney, etc).

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled “Registered report: Biomechanical remodeling of the microenvironment by stromal Cav1 favors tumor invasion and metastasis” for further consideration at eLife. Your revised article has been favorably evaluated by Fiona Watt (Senior editor), a member of the Board of Reviewing Editors, and three reviewers. The manuscript has been improved but there are a few minor remaining issues that need to be addressed before acceptance, as outlined below:

Both Abstract and Introduction should clearly state, as it was stated in the first version of this manuscript, that the 50 papers being replicated are the top 50 most impactful ones in the cancer biology field, and not just 50 random papers from this field. This critical piece of information has now been removed from both Abstract and Introduction. Also, the Abstract should state clearly that the authors are replicating not the whole paper, but only a fraction of it.

Several important controls are missing: it would be important to check by WB that the pMEFs generated are actually KO for caveolin-1. Also, to make sure that WT pMEFs have increased expression of (at least) smooth muscle actin (SMA) compared to KO pMEFs, as a marker of increased activation and ECM remodeling capabilities of these pMEFs.

If the authors wish to insist on the discrepancy between the Capozza et al. and the Goetz et al. studies, they should inject the B16F10 cells used by Capozza to clarify whether the discrepancy may be caused by a difference in cell lines.

Regarding statistics, the reviewers were confused by the answer of the authors to question 3 of Reviewer #4. If the authors want to see if the effect of these distinct groupings is equivalent, it seems one should not do a superiority power calculation but rather a bioequivalence. The authors have used a 15% adjustment, which is what is done to convert numbers in a parametric calculation to a non-parametric calculation. The answer seems to imply (in protocol 2) that the authors found they needed 39 animals and adjusted this to 48). Then the authors used the sample size to see what effect size this would have 80% power to detect? However, the reviewers did not follow how the authors got to 39 animals in the first place. Could the authors clarify this point?

Finally, the authors should indeed report also the raw p-values. This is very important because corrected p-values (especially with Bonferroni) will always be less significant than the raw ones, so some of the significant results could be “lost” if only corrected p-values were reported.

The authors should provide a more extensive and more balanced discussion of the literature on the function of caveolin-1 in different cancers; some missed references are listed in the comments of reviewer 1 below. Importantly, the Capozza et al. paper, presented as in contradiction with the findings by Goetz et al., may not be in contradiction since Capozza et al. focused on the size of the primary tumours whereas Goetz et al. focused on metastasis (see also comments from Reviewer 1 below for more details). This should be clarified. If the authors wish to insist on a discrepancy between the Capozza et al. and the Goetz et al. studies, it would be a good idea to inject the B16F10 cells used by Capozza et al. (Reviewer 3's suggestion), to clarify whether the discrepancy may be caused by a difference in cell lines.

We have included a more balanced discussion of the literature on caveolin-1, including the references in the comments of Reviewer #1. And we have removed the indicated discrepancy between the Capozza et al. and the Goetz et al. studies and included the results of the Goetz et al., Capozza et al., and Chang et al. studies.

There are a number of experimental points listed in the reviews comments appended below that need to be clarified.

Extracts from Reviewer #1:

A significant overstatement made by the authors of this Reproducibility Project pertains to protocol 2. They claim that there is a contradiction between the tested paper (Goetz et al. Cell 2011) and another one by the Lisanti group: “In contrast to these findings, Capozzo and colleagues showed that intradermal coinjection of nude mice with B16F10 melanoma cells and Cav1 KO neonatal dermal fibroblasts increased primary tumor growth when compared to coinjection of tumor cells with WT fibroblasts ().” We disagree that there is conflict here, since the original paper by Goetz et al. was focused on studying changes in stromal remodeling and, as a consequence of them, correlative changes in tumor local invasion and distant metastasis. The growth of the primary tumor was not the main focus of Goetz et al., and hence it was not as critically evaluated as ECM remodeling or metastasis, as it was the case for Capozza et al. Due to the nature of the allograft/xenograft experiments of Figure 7, focused on evaluating ECM remodeling and the appearance of metastasis, it was also possible to measure the size of the primary tumor, but this was not as critically monitored as the metastasis formation. Hence, we believe that it is not correct to claim that one of the main conclusions of the original paper is that the growth of the primary tumor is not affected by the absence of Cav1 expression in fibroblasts. Therefore, we believe is not correct to include primary tumor growth as one of the statistical analysis to be performed in protocol 2. In the experimental approach that Fiering et al. have chosen, the primary tumor growth was not significantly different between any of the studied conditions. However, primary tumor growth was decreased in the absence of Cav1 expression in mammary gland allografts (Figure 7A and S7A) and in mammary gland xenografts (Figure 7B and S7B). It is important to note that in these approaches, the whole mammary gland was deficient for Cav1 expression, not only the fibroblasts, and hence they are not exactly comparable. In the same regard, Capozza et al. used neonatal dermal fibroblasts, which are not exactly the same as MEFs. Further, Fiering et al. fail to cite other papers that apparently contradict Capozza et al. and are in line with results of Figure S7A and S7B. One example comes from the Dvorak group, which showed that subcutaneous injection of B16 melanoma cells in Cav1 KO mice leads to reduced tumor growth compared to injection of tumor cells in WT mice (Chang et al., Am J Path 2009). Fiering et al. should also cite this work, but, even more importantly, these differences highlight the complexity of this biological problem, and the potential difficulty to reproduce observations made in several labs.

In the particular case of the subcutaneous xenograft model (Figure 7C), the main conclusions to be reproduced should thus be:

1) Absence of Cav1 expression in fibroblasts inhibits metastasis of LM-4175 breast tumor cells.

2) Absence of Cav1 expression in fibroblasts results in an less anisotropic ECM (with a lower proportion of parallel Fibronectin fibers).

3) A statistical significant correlation between the anisotropy of the intratumoral fibers and the total number of metastasis per mouse (Spearman's rho correlation).

4) Presence or absence of Cav1 expression in fibroblast modifies cell shape (elliptical factor of SMA+ cells).

We have included a more balanced discussion of the literature on caveolin-1. And we have removed the indicated discrepancy between the Capozza et al. and the Goetz et al. studies and included the results of the Goetz et al., Capozza et al., and Chang et al. studies as suggested. We also try to focus on the experiments being replicated and any direct replications of them. We agree that the metastasis formation in this model is the primary focus of Protocol 2 and have determined the appropriate sample size to have adequate power for this analysis. As an added aspect of the experimental approach, we have included the primary tumor growth data analysis, just like the original study, with the effect size we would be able to detect, if there is one, with the sample size we are using. The inclusion of this data is also only in respect to this exact model, not to the potential of Cav1 on primary tumor growth in other models, including the ones presented in Goetz et al. (Figures S7A-B). As such, we will restrict our analysis to the experiments being replicated and will not include discussion of experiments not being replicated in this study.

[…] Therefore, the authors of this Reproducibility Project should undertake a comprehensive review of the literature on this subject. In the current version they have only chosen those papers that somehow contradict the original one, but they do not mention to what extent they are different (type of tumor, TMA vs. mouse experiments, etc.) and missed important literature that should be included, some of which is consistent with Goetz et al.

We have included a more balanced discussion of the literature on caveolin-1. Additionally, we have removed the indicated discrepancy between the Capozza et al. and the Goetz et al. studies and included the results of the Goetz et al., Capozza et al., and Chang et al. studies as suggested as well as include the other suggested references. We try to focus on the experiments being replicated and any direct replications of them, instead of a comprehensive review of all the literature on this subject, which includes many conceptual or related experiments, which would be more appropriate for a review.

Are the proposed experiments appropriately designed? The proposed experiment is appropriately designed, although several small modifications should be highlighted:

1) Protocol 1, procedure points 5-6 do not reflect the original protocol and should be indicated by an asterisk.

We have reached out the original authors to clarify the exact protocol that was used. The dissociation of the pMEFs by trypsin was performed in the original study, but we have revised the other steps to reflect the exact protocol used.

2) Protocol 2, it must clearly indicate that the MEFs co-injected are primary MEFs.

We have made this clearer and corrected any references to MEFs as primary MEFs.

3) Protocol 2, procedure point 3b. Nude mice do not require shaving since they do not have any hair; shaving will thus induce unnecessary skin irritation, which could lead to stromal activation.

Thank you for catching this. We have removed this from the procedure.

4) Protocol 2, procedure point 3d. It is important not to take the needle out of the skin till the matrigel has gelified. Gelification takes very short time at body temperature (around a minute).

Thank you for the additional details. We have added this into the procedure.

5) Protocol 2, procedure point 6. Although in the original study the final analysis point was set at 70 days, it is important to keep some kind of flexibility for this end-point. The investigators must consider shortening this time if the number of mice euthanized compromise the success of the whole study (due to the reasons explained in point 5).

Thank you for this information. We have included a statement reflecting the potential of shorting the study time to ensure enough mice are obtained for analysis. If the time is changed because of an increase in having to euthanize mice due to complications, the change in timeframe will be recorded.

6) Protocol 3, sampling. Please stain at least 2 sections per tumor, since some sections are difficult to evaluate due to necrosis, autofluorescence or damage during the staining procedure.

We have updated the protocol to reflect 2 sections per tumor. We have also included the exclusion criteria outlined here that might prevent a section or image from being included in the analysis.

7) Protocol 3, materials. The authors plan to use Alexa488 conjugated donkey anti-rabbit IgG (although later in procedure point 6, they claim to use goat-anti rabbit FITC, please check this inconsistency). In the original study, goat-anti rabbit Cy3 was used. This selection is not trivial, since the plasmid used to express Luciferase in the LM-4175 cells (HSV-tk1-GFP-luc) contains an IRES-GFP sequence. Thus, LM-4175 cells are green (although not really bright), and therefore this fluorescence channel must not be used for additional staining.

Thank you for catching this. We have changed the Alexa488 to Alexa594 so the imaging is not in the same channel as the GFP from the integrated luciferase vector in the LM-4175 cells.

8) Protocol 3, materials. The authors plan to use Alexa647 conjugated donkey anti-mouse IgG as we have done. However, later in procedure section, point 6, they claim to use goat-anti mouse CF640R, please check this inconsistency.

Thank you for catching this. We have corrected this to be consistent.

Are the proposed statistical analyses rigorous and appropriate? The use of Wilcoxon-Mann-Whitney test to compare total metastatic foci counts in the three groups is well sounded and agrees with the statistical analysis from the original paper; however, since the original paper did report raw p-values only, we believe it would be more appropriate to report also non-corrected p-values in the reviewed paper. On top of that, post-hoc methods based on the control of the FWER (such as Bonferroni) tend to be too conservative compared to methods controlling the FDR, which are the ones we believe should be used by Fiering et al. The use of Bonferroni would significantly dampen the probability to find significant differences, which we believe is somehow unfair for this Reproducibility Project.

We have included the uncorrected tests in the analysis plan of Protocol 2 and 3. However, we do feel the use of Bonferroni to correct for multiple comparisons is appropriate. We have used an appropriately adjusted alpha error in our power calculations to account for this as these are a priori comparisons. And while FDR control provides the same degree of assurance as Bonferroni correction that there is indeed some effect, FDR is not useful to be certain that any single significant result is accurate, since some false positives are allowed, and thus is why we use a technique like Bonferroni correction, which provides a conservation control of the FWER.

On the other hand, Fiering et al. report that they will perform a meta-analysis with the results of the original paper and the new results, but there are no details about the methodology used for such a meta-analysis.

We have included in the analysis plan that we will be combining the original and replication effect sizes using a random-effects meta-analytic approach.

What can the replication team do to maximize the quality of the replication?

Please see the modifications of the protocol highlighted above. Although with 3 regions imaged per sections the authors claim to obtain a minimum power of 80%, some tumors are difficult to image (necrotic damage, autofluorescence, low fibronectin deposition or damage due to preparation or manipulation). For these reasons, we would highly recommend to stain at least two sections per tumor and to image at least five regions per section, although the more the better.

We have updated the protocol to reflect 2 sections per tumor and 5 regions per section. We have also included the exclusion criteria outlined here that might prevent a section or image from being included in the analysis.

Extracts from Reviewer #2:

Several points not specified in should be clarified with the original authors, before initiating the experiments. eLife has verified these points with the original authors and the responses follow the questions:

The order code of the nude mice and their age range when used for the study.

Nude mice, official name from Harlan: Athymic Nude Mouse -Hsd: Athymic Nude-Foxn1.

This is the order code of the nude mice in the manuscript.

And their age range when used for the study: 8-10 weeks when they arrive, about 10-12 when experiment was done.

We have updated the language to reflect the age of mice when they arrive vs. the age when the mice are injected.

Whether DMEM with high glucose was used for in vitro culture of MEFs. Normal DMEM, not high glucose.

We have updated the Materials and Reagents section to reflect this.

Original matrigel stock concentration, so the used relative dilution yields the same final concentration for implantation.

Typical protein concentrations for BD Matrigel Matrix are between 9-12 mg/ml.

This is the same Matrigel Matrix with the same concentration range listed in the Materials and Reagents section of Protocol 2. The Manufacturer is Corning, but is the same catalog and formulation as BD.

Protocol 2:

For metastasis detection, a range of exposure times from 20s to 2 min was used to detect both large and small metastases. Thus, each mouse and excised organ should be assessed to reach maximum sensitivity and count a maximum of micrometastases. To reach high sensitivity, when monitoring the lungs, liver and brain, the lower abdomen (containing tumor and luminescence in the bladder) should be shielded during longer exposure times. Also, for metastasis detection of excised organs, 2 min should be included as longest exposure time.

The maximum exposure time of 2 min is included in the procedure. The original paper only took 0.2, 1, 20, and 60 sec exposures, but we agree that increasing the potential exposure time to 2 min will enable maximum sensitivity to count micro-metastases. In correspondence with the authors the original study used only the ex vivo imaging on organs to determine the metastatic foci count. The in vivo imaging was used for assessing tumor growth and metastasis onset only.

Protocol 3:

Irrespective of the power calculation, because of expectable intra-tumor variation of stromal density and organization, several sections from the same tumor should be analyzed for fibronectin alignment and cell elongation. The analysis of only 3 fields from a single section may be flawed by accidental sample variability and insufficient representativity.

We have updated the protocol to reflect 2 sections per tumor and 5 regions per section. We have also included the exclusion criteria outlined by Reviewer #1 that might prevent a section or image from being included in the analysis.

Extracts from Reviewer #4:

For protocol 2 and 3 the exact details of what was found in the original paper are reported (generally groups means and SDs) so I easily can follow this logic of how the sample size/power calculations have been derived. I have a number of questions regarding assumptions made in these sample size calculations:

1) Often the SD used in the power calc is estimated on a group of size between 6 and 15 so these estimates will tend to be underestimates. This is not so much of a problem when the effect sizes are approximately 1 SD but it could still be taken account of. I cannot replicate the pooled SDs used in the tables – mine come out slightly larger.

We have checked our calculations, and obtained the same pooled SDs as originally reported. We are using the formula:

2) Sometimes the type 1 error is adjusted for multiple testing and sometimes it is not. If the same animals are studied for several outcomes then these should be treated as multiple testing problems. When several organ sites are examined this must surely be the same animals?

Thank you for catching this oversight. We performed the calculations with the alpha error of the multiple organs at 0.01 (to account for the 6 groups) instead of 0.05. As expected, the sample size we are using is not sufficient to perform the individual organ comparisons that were performed in the original paper. We have excluded these from our analysis. However, the main claim is the comparisons between the total metastatic foci count per mouse, which correctly has the alpha error adjusted.

3) It seems odd to me that the replication study wants to replicate results that were not statistically significant in the original study? In these cases they argue that they select an effect size that they have 80% power to detect.

Sometimes we are including comparisons that did not obtain a statistically significant effect in the original study to determine if we see the same non-statistically significant effect size, and because some of these effects, such as the comparison between LM-4175 cells alone and LM-4175 cells with KO MEFs, are of scientific interest to determine if they are not the same. The effect size we report is determined by the sample size, which is determined by other statistically significant effects, the alpha error, and the pre-defined power of 0.80. Thus, we are presenting what effect size could be detected, if there is one, for these comparisons.

4) There are many hypotheses being tested in this replication. Are they all equally important? Are some of them nested? If half of the tests are statistically significant will this be regarded as a validation of the original report? I am not confident that these sample sizes will have sufficient power for so many repeated tests.

We have now excluded all of the individual organ comparisons that were performed in the original paper, because our sample size is not sufficient. However, the main claim is the comparisons between the total metastatic foci count per mouse, which correctly has the alpha error adjusted. With the rest of the included tests, we have performed sample size calculations to ensure our sample size is sufficient with a power of at least 80%.

5) There is some confusion in statistical language which may be a feature of the G*power program. The sample size calculations state that t-tests (to detect standardised effects) will be used but the analysis plan uses non-parametric tests (Kruskall Wallis and Mann Whitney, etc).

Yes, we agree that the language can be a little confusing, however, the Kruskall Wallis and Mann Whitney are listed in the sample size calculations as the type of F, or t, non-parametric test used, similar to how Spearman’s rho is listed as a type of correlation test. G*Power lists all parametric and non-parametric tests within the same test family.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Both Abstract and Introduction should clearly state, as it was stated in the first version of this manuscript, that the 50 papers being replicated are the top 50 most impactful ones in the cancer biology field, and not just 50 random papers from this field. This critical piece of information has now been removed from both Abstract and Introduction. Also, the Abstract should state clearly that the authors are replicating not the whole paper, but only a fraction of it.

The first two sentences of the Abstract, as well as the last sentence was changed by an editorial revision on 10/23/14, with the same three sentences used to ensure consistency across the Registered Reports. This is the same language used in the published Registered Reports to date. However, if the reviewers prefer the language to include the word ‘impactful’, we defer to the eLife editorial board who can comment on the ability to do this for the published Registered Reports to ensure consistency.

We have also added the exemplifier ‘which are a subset of all the experiments reported in the original publication’ to the Abstract when describing the experiments outlined in the Registered Report.

Several important controls are missing: it would be important to check by WB that the pMEFs generated are actually KO for caveolin-1. Also, to make sure that WT pMEFs have increased expression of (at least) smooth muscle actin (SMA) compared to KO pMEFs, as a marker of increased activation and ECM remodeling capabilities of these pMEFs.

We agree and have updated the manuscript to include these quality controls, which were similarly reported in Goetz et al. in Figures 7CA and Supplemental Figure 2A.

If the authors wish to insist on the discrepancy between the Capozza et al. and the Goetz et al. studies, they should inject the B16F10 cells used by Capozza to clarify whether the discrepancy may be caused by a difference in cell lines.

We have edited the text in the Introduction to remove any indication of a discrepancy between the Capozza et al. and the Goetz et al. studies. We agree that the metastasis formation in this model is the primary focus of Protocol 2 and have indicated this in the Introduction as well. As an added aspect of the experimental approach, we have included the primary tumor growth data analysis, just like the original study, however the inclusion of this data is also only in respect to this exact model, not to the potential of Cav1 on primary tumor growth in other models, including the ones presented in Goetz et al. (Figures S7A-B). As such, we will restrict our analysis to the experiments being replicated and will not include discussion of experiments not being replicated in this study.

Regarding statistics, the reviewers were confused by the answer of the authors to question 3 of Reviewer #4. If the authors want to see if the effect of these distinct groupings is equivalent, it seems one should not do a superiority power calculation but rather a bioequivalence. The authors have used a 15% adjustment, which is what is done to convert numbers in a parametric calculation to a non-parametric calculation. The answer seems to imply (in protocol 2) that the authors found they needed 39 animals and adjusted this to 48). Then the authors used the sample size to see what effect size this would have 80% power to detect? However, the reviewers did not follow how the authors got to 39 animals in the first place. Could the authors clarify this point?

To clarify the earlier comments and the intent of these calculations, the detectable effect size reported for the primary tumor size analysis is not to test if the groups are equilavent, but rather to determine what effect size can be detected if there is a difference. The original study did not observe a statistically significant effect based on the sample size they used, and as the reviewer points out, this does not imply the groups are equivalent, but rather not different. The same analysis will be conducted in the replication study, but since the ability to detect a statistically significant difference is dependent on sample size we calculated the effect size that could be detected with the sample size that will be used.

To clarify the sensitivity calculation, we knew the sample size is 49 based on the total metastatic foci per mouse sample size calculation. However, since we will be performing a non-parametric test, we adjusted the sample size accordingly since we were determining the detectable effect size using a parametric calculation. We have updated this section to clarify this point. We also adjusted the numbers to reflect the detectable effect size using the average group size since 41 (or 49) are not multiples of the number of groups, which is 3. Originally we had used the next lowest sample size divisible by 3, which was 39 and 48, respectively, but since this is an estimate of the detectable effect size, the difference does not change the analysis plan. This calculation is intended to report what effect size can be detected with the sample size and test used, not the sample size needed to detect a given effect size.

Finally, the authors should indeed report also the raw p-values. This is very important because corrected p-values (especially with Bonferroni) will always be less significant than the raw ones, so some of the significant results could be “lost” if only corrected p-values were reported.

We will report both corrected and uncorrected p-values. We will also report the effect size and 95% confidence interval to provide another means of evaluating the data.

Reagent	Type	Manufacturer	Catalog #	Comments
6–8 week old B6129SF2/J mice (Cav1 WT)	Animal model	Jackson laboratory	101045	1 male and 3 females for breeding
6–8 week old CAV1<tm1mls>/J mice (Cav1 KO)	Animal model	Jackson laboratory	004585	1 male and 3 females for breeding
Ethanol	Chemical	Sigma–Aldrich	E7023	Original not specified
PBS, without MgCl2 and CaCl2	Buffer	Sigma–Aldrich	D8537	Original not specified
0.05% trypsin/0.48 mM EDTA	Cell culture	Sigma–Aldrich	T3924	Original not specified
50 ml tubes	Labware	Sigma–Aldrich	CLS430290	Original not specified
Dulbecco's modified Eagle's medium—low glucose, without L-glutamine	Cell culture	Sigma–Aldrich	D5546	Original not specified
Fetal bovine serum	Cell culture	Sigma–Aldrich	F0392	Original not specified
L-glutamine	Cell culture	Sigma–Aldrich	G7513	Original not specified
100× Pen/Strep	Cell culture	Sigma–Aldrich	P4333	Original not specified
150 mm tissue culture dishes	Labware	Sigma–Aldrich	CLS430599	Original not specified
100 mm tissue culture dishes	Labware	Sigma–Aldrich	CLS430167	Original not specified
60 mm tissue culture dishes	Labware	Sigma–Aldrich	CLS430166	Original not specified

Reagent	Type	Manufacturer	Catalog #	Comments
60 mm tissue culture dishes	Labware	Sigma–Aldrich	CLS430166
PBS, without MgCl2 and CaCl2	Buffer	Sigma–Aldrich	D8537	Original not specified
RIPA lysis buffer	Buffer	Specific brand information will be left up to the discretion of the replicating lab and recorded later
Cell scraper	Labware
Refrigerated centrifuge	Equipment
Bradford assay	Reporter assay
Molecular weight marker	Western materials
6× SDS-PAGE sample buffer	Buffer	Specific brand information used to make these reagents will be left up to the discretion of the replication lab and recorded later
SDS-PAGE gel	Western materials
Tris-glycine SDS-PAGE running buffer	Buffer
Electrotransfer buffer	Buffer
Ponceau S stain	Stain
TBS buffer	Buffer
PVDF membrane	Western materials	Specific brand information will be left up to the discretion of the replicating lab and recorded later
Tween-20	Chemical
Non-fat dry milk	Western materials
ECL chemiluminescent reagent	Western materials
Mouse anti-Cav1 (clone 2297)	Antibodies	BD Biosciences	610406	Original clone/catalog# unspecified
Mouse anti-α-smooth muscle actin (clone 1A4)	Antibodies	Sigma–Aldrich	A5228
Mouse anti-γ-tubulin (clone GTU-88)	Antibodies	Sigma–Aldrich	T6557	Original clone/catalog # unspecified
Goat anti-mouse-HRP	Antibodies	Life Sciences	32,430

Reagent	Type	Manufacturer	Catalog #	Comments
LM-4175 cells expressing HSV-tk1-GFP-Fluc	Cell line	Original lab	n/a	From original lab
Dulbecco's modified Eagle's medium—low glucose, without L-glutamine	Cell culture	Sigma–Aldrich	D5546	Original not specified
PBS, without MgCl2 and CaCl2	Buffer	Sigma–Aldrich	D8537	Original not specified
0.05% trypsin/0.48 mM EDTA	Cell culture	Sigma–Aldrich	T3924	Original not specified
50 ml tubes	Labware	Sigma–Aldrich	CLS430290	Original not specified
Fetal bovine serum	Cell culture	Sigma–Aldrich	F0392	Original not specified
L-glutamine	Cell culture	Sigma–Aldrich	G7513	Original not specified
100× pen/Strep	Cell culture	Sigma–Aldrich	P4333	Original not specified
100 mm tissue culture dishes	Labware	Sigma–Aldrich	CLS430167	Original not specified
60 mm tissue culture dishes	Labware	Sigma–Aldrich	CLS430166	Original not specified
Matrigel matrix	Cell culture	Corning	356234	Original from Becton Dickinson
8–10 week old female athymic nude mice	Animal model	Harlan	Hsd:Athymic Nude-Foxn1nu	Mice should be acclimated for 2 weeks before the start of experiment
Ketamine	Chemical	Specific brand information will be left up to the discretion of the replicating lab and recorded later
Xylazine	Chemical
25 G needle	Labware	Sigma–Aldrich	Z192406	Original not specified
1 ml syringe	Labware	Sigma–Aldrich	Z192090	Original not specified
VivoGlo Luciferin	Reporter assay	Promega	P1042	Original not specified
IVIS Imaging System	Instrument	PerkinElmer	200 Series
Living Image software	Software	PerkinElmer	Version 4.3.1
O.C.T. compound (Tissue-Tek)	Buffer	VWR	25,608-930	Original not specified

Reagent	Type	Manufacturer	Catalog #	Comments
Fluoroshield	Chemical	Sigma–Aldrich	F6182	Included during communication with original authors. Original lab used Permafluor.
Acetone	Chemical	Specific brand information will be left up to the discretion of the replicating lab and recorded later
Chloroform	Chemical
PBS, without MgCl2 and CaCl2	Buffer	Sigma–Aldrich	D8537	Originally not specified
Bovine serum albumin	Chemical	Sigma–Aldrich	A9647	Originally not specified
Rabbit IgG isotype control	Antibodies	Sigma–Aldrich	I5006	Originally not specified
Rabbit anti-fibronectin	Antibodies	Sigma–Aldrich	F3648	Stock = 0.5–0.7 mg/ml; Dilute 1:200
Mouse IgG2a isotype control	Antibodies	Sigma–Aldrich	M5409	Originally not specified
Mouse anti-α-smooth muscle actin	Antibodies	Sigma–Aldrich	A5228	Stock = ∼2 mg/ml; Dilute 1:100
Alexa 594 conjugated donkey anti-rabbit IgG	Antibodies	Jackson Immuno Research	711-545-152	Original lab used goat-anti-rabbit-Cy3
Alexa 647 conjugated donkey anti-mouse IgG	Antibodies	Jackson Immuno Research	715-605-151	Originally not specified
Hoechst stain 33,258	Stain	Sigma–Aldrich	14,530	Dilute 1:1000
Confocal microscope	Instrument	Zeiss	LSM510	Original was Leica SPE (communication with original authors)
Image acquisition software	Software	Zeiss	ZEN 2009	Originally not specified
Metamorph microscopy automation and imaging analysis software	Software	Molecular Devices	6.2r1
Excel	Software	Microsoft

Dataset being analyzed	Mean	SD	N
LM-4175 only	2.50	1.975	6
LM-4175 + Cav1 WT pMEFs	28.42	22.24	12
LM-4175 + Cav1 KO pMEFs	11.67	11.10	15

Group 1	Group 2	Pooled SD	Effect size d	A priori power	Group 1 sample size	Group 2 sample size
LM-4175 only	LM-4175 + Cav1 WT pMEFs	18.47	1.403155	80.7%	7	21
LM-4174 only	LM-4175 + Cav1 KO pMEFs	9.58	1.390827*	80.0%*	7	21
LM-4175 + Cav1 WT pMEFs	LM-4175 + Cav1 KO pMEFs	16.93	0.989463	81.9%	21	21

A sensitivity calculation was performed since the original data showed a non-significant effect. This is the effect size that can be detected with 80% power.

Dataset being analyzed	Mean	SD	N
LM-4175 only	2.337 × 1010	1.856 × 1010	6
LM-4175 + Cav1 WT pMEFs	2.825 × 1010	3.901 × 1010	13
LM-4175 + Cav1 KO pMEFs	2.312 × 1010	1.368 × 1010	15

Kruskal–Wallis statistic	p-value
0.8878	0.6415

Groups	Effect size f	A priori power	Total sample size
LM-4175 only, LM-4175 + Cav1 WT pMEFs, LM-4175 + Cav1 KO pMEFs	0.504525*	80.0%*	41† (3 groups)

A sensitivity calculation was performed since the original data showed a non-significant effect. This is the effect size that can be detected with the sample size reported and 80% power.

Since the non-parametric Kruskal–Wallis test will be performed for the analysis instead of an ANOVA, the sensitivity calculation was performed with a ∼15% adjustment in sample size to calculate the effect size that can be detected with 80% power. The total sample size of 49, which comes from the total metastatic foci per mouse sample size calculation, was reduced by ∼15%–41 for this calculation to estimate the detectable effect size.

Dataset being analyzed	N	Mean	SEM	SD
LM-4175 only	5	36.8	0.7	1.565
LM-4175 + Cav1 WT pMEFs	8	50.3	2.3	6.505
LM-4175 + Cav1 KO pMEFs	10	41.5	1.1	3.479

Group 1	Group 2	Pooled SD	Effect size d	A priori power	Group 1 sample size	Group 2 sample size
LM-4175 only	LM-4175 + Cav1 WT pMEFs	5.27	2.559575	82.7%*	4	4*
LM-4175 + Cav1 WT pMEFs	LM-4175 + Cav1 KO pMEFs	5.03	1.748808	83.1%	7	7

7 tumors will be used based on the WT vs KO comparison making the power 94.0%.

Dataset being analyzed	N	Mean	SEM	SD
LM-4175 only	224	1.70	0.03	0.449
LM-4175 + Cav1 WT pMEFs	1246	2.14	0.03	1.059
LM-4175 + Cav1 KO pMEFs	763	1.68	0.02	0.5524

Group 1	Group 2	Pooled SD	Effect size d	A priori power	Group 1 sample size	Group 2 sample size
LM-4175 only	LM-4175 + Cav1 WT pMEFs	0.99	0.444072	80.3%	85	85
LM-4175 + Cav1 WT pMEFs	LM-4175 + Cav1 KO pMEFs	0.90	0.510625	80.6%	65	65

Dataset being analyzed	N	Mean	SEM	SD
LM-4175 only	5	33.1	4	8.944
LM-4175 + Cav1 WT pMEFs	8	51.3	2	5.657
LM-4175 + Cav1 KO pMEFs	10	35.3	2	6.325

Group 1	Group 2	Pooled SD	Effect size d	A priori power	Group 1 sample size	Group 2 sample size
LM-4175 only	LM-4175 + Cav1 WT pMEFs	7.03	2.588043	83.5%*	4	4*
LM-4175 + Cav1 WT pMEFs	LM-4175 + Cav1 KO pMEFs	6.04	2.648198	85.0%†	4†	4†

7 tumors will be used based on the fibronectin fiber orientation analysis making the power 94.4%.

7 tumors will be used based on the fibronectin fiber orientation analysis making the power 99.3%.

Groups	Number of simulations	A priori power	Total sample size
% fibronectin fibers within ± 20° and number of metastasis	10,000*	83.1%	10

The shared data from XY pairs was randomly sampled from, with replacement, to create simulated data sets with preserved correlated structure. For a given n (the number of observations) 10,000 simulations were run and Spearman's rho was calculated for each simulated data set. The power was then calculated by counting the number of times p ≤ 0.05 and dividing by 10,000.