Statement of Work (CRC Response Testing).docx

Appendix “B”

Argonne National Laboratory Statement of Work Title: Pilot Drug Screening Study for IMPROVE Date: August 4, 2023

1.0 Background

The project of Innovative Methodologies and New Data for Predictive Oncology Model Evaluation (IMPROVE) is a collaborative research effort of the Argonne National Laboratory and the National Cancer Institute. The goal of IMPROVE is to build a framework to evaluate and compare computational models that predict anti-cancer drug responses. One of the aims of IMPROVE is to generate new large-scale drug screening data on patient-derived cancer models, which can be used for evaluating drug response prediction models. The data generation process is divided into two phases, the pilot phase and the large-scale screening phase. The size of pilot drug screening studies will be much smaller than the size of large-scale screening. The purpose of pilot studies is mainly to evaluate the qualification of a drug screening vendor for the large-scale study.

2.0 Scope

The Argonne National Laboratory (Argonne) will request The Contractor to perform a pilot drug screening study for the IMPROVE project.

3.0 Objectives

At the request of Argonne, The Contractor will screen 100 compounds in 7 colorectal cancer (CRC) models including 2 cancer cell lines and 5 patient-derived organoid (PDO) models. Data generated through the experiments will be provided to Argonne. The generated data will be used to evaluate (1) the qualification of The Contractor as a potential vendor for a large-scale drug screening study in near future and (2) the feasibility of using the data for evaluating existing drug response prediction models.

4.0 Tasks and Study Setup/Assumptions

The Contractor will screen 100 compounds in 7 CRC models including 2 cancer cell lines and 5 PDO models. For the 2 cancer cell lines, which are HT-29 and SW620, The Contractor will use their inhouse specimens. For PDO specimens, Argonne will purchase and ship them to The Contractor. The list of compounds and the dose range to be screened will be provided by Argonne and can be modified based on inputs from The Contractor. The Contractor will purchase the compounds specific to this screening study. The compound purchase cost will be charged to Argonne. The Contractor is responsible for in accordance with all laws and guidelines the acceptance, storage, usage, and disposal of wastes containing chemotherapeutic and/or general pharmaceutical agents and remaining biospecimen material. The following is the study setup.

Phase 1) Testing 100 compounds in two CRC cell lines Aim: To test a 7-point dose range of 100 compounds in two CRC cell lines cultured in 3D.

Setup: CRC cell line models SW620 and HT-29 will be seeded in a hydrogel in 384-wells plates. After medium addition, the cell lines will be pre-cultured for 2 days. Subsequently, the seeded cells will be exposed to a 7-point dose range of 100 compounds in triplicates for 3 days. Controls on each screening plate: DMSO and/or PBS (negative control; n=8 for each type of control; concentration equivalent to compound solvent), a 7-point dose range of one standard of care (SoC) compound (t.b.d.) in singlicate (plate control), no fetal bovine serum (from day 0: growth control; n=4), and 1μM Staurosporine (positive control; n=8). The cells will be fixed to mark the actin cytoskeleton with Rhodamine Phalloidin and the nuclei with Hoechst for high content imaging (HCI). The stained cell structures will subsequently be processed for image-based analysis in the 3D analysis platform Ominer® to assess cell viability and the effect of the compounds on the outgrowth and morphology of the tumoroids by multiparametric analysis.

Assay size: 2400 wells (7-point dose range, triplicates at each dose) per model in 2 cell line models.

Expected assay performance criteria:

- The cell lines (1500 cells per well) grow out to multicellular structures in the 384-wells plates in the negative control conditions and growth will be compromised upon Staurosporine treatment (p<0.05).

Phase 2) Testing 100 compounds in 5 CRC organoid models Aim: To test a 7-point dose range of 100 compounds in 5 CRC organoid models*.

Setup: More than 5 CRC PDO models will be shipped to The Contractor. Upon arrival, the first five models will be expanded between 1 and 5 passages and biobanked. If a model does not grow, it will be replaced by another model from the shipped selection. The pre-cultured set of 5 models will subsequently be seeded in a hydrogel in 384-wells plates. After medium addition, the organoid models will be allowed to grow out for two days. On day three the medium will be refreshed, and the organoids will be exposed to a 7-point dose range of 100 compounds in triplicates for 3 days. Controls on each screening plate: DMSO and/or PBS (negative control; n=8 for each type of control; concentration equivalent to compound solvent), 0ng/ml epithelial growth factor (from day 0: growth control; n=4), a 7-point dose range of one standard of care (SoC) compound (same as Phase 1) in singlicate (plate control), and 1μM Staurosporine (positive control, n=8). The organoids will be fixed to mark the actin cytoskeleton with Rhodamine Phalloidin and the nuclei with Hoechst. The stained organoids will subsequently be processed for image-based analysis in the 3D analysis platform Ominer®.

Assay size: 2400 wells (7-point dose range, triplicates at each dose) per model in 5 organoid models.

Expected assay performance criteria:

- The organoid models (300 organoids per well) grow out to multicellular structures in the 384-wells plates in the negative control conditions and growth will be compromised upon Staurosporine treatment (p<0.05).

* The selection of CRC organoids will be made from the Human Cancer Models Initiative (HCMI) collection provided by the American Type Culture Collection (ATCC). The timeline and the number of models that can be expanded sufficiently to perform the experiments depend on the model growth rates. A model that does not grow will be discontinued and replaced by the next one on the list.

5.0 Delivery

The Contractor will generate the following and deliver them to Argonne.

a. A report in PowerPoint format will be generated for each (sub) phase and digitally delivered. Contents include experiment setup/conditions, quality control, and the imaging-based readouts, example images of the organoid model and the effect of the treatments, quantification and graphical interpretation of treatment effects on the organoids but will not include detailed protocols or medium formulations.

b. Measurements of the organoid size, nucleus count per organoid, nucleus size, fraction of dead cells per organoid, and other relevant features related to the morphology of the organoids and their lumen will be provided. Raw data of morphological features will be shared as separate .csv files.

c. Complete raw data will be available for transfer to Argonne including raw images.

d. Updates on progress will be provided on a regular (per 2-week) basis by email (brief summaries).

e. Upon project completion, (1) surplus compounds (stocks) purchased specific for this study, (2) frozen cryovials of PDO specimens provided by Argonne but not used in the study, and (3) leftover PDO cultures from the drug screening study will be shipped to Argonne and/or stored in The Contractor’s inventory for an annual fee at Argonne’s choice.

f. Details of experimental procedure for publication purposes will be provided at the end of the project, including references and others.

6.0 Government-Furnished Property

Argonne will purchase ATCC HCMI PDO specimens, in the form of cryopreserved organoids, and ship them to The Contractor. One cryovial of 1 x 106 cells per model. The Contractor will not use the specimens for any purpose not included in the drug screening study requested by Argonne. Argonne may choose to supply more than 5 PDO models as backups in case of failure in establishing cell models.

7.0 Place of Performance

The whole study will be accomplished at The Contractor's facility.

8.0 Period of Performance