Frequently Asked Questions

Sample Procurement

Are the samples sourced from commercial vendors?

Yes, we procure live tissue samples from commercial vendors who in turn source them from a variety of hospitals. We also use the NCI network hospitals to obtain tissues.

Is clinical data associated with these samples available?

We do have some clinical data available including therapeutic history from de-identified patient samples.

Biobank

Are the tumoroids frozen, separated and then reused?

Yes, we biobank them for the purpose of repeatability. Additionally, We have demonstrated growth of up to five generations without issue.

Are the PBMC’s cryopreserved as such; or, do you maintain them as T-cells?

The entirety of the PBMCs are stored without expansion in lot sizes sufficient for long-term, longitudinal and repeat studies.

Changes Over Time

Do the PDTs change over time?

We see no changes up to 5 generations. We also do not typically exceed a 28-day study timeframes in our experiments to ensure there are no substantive changes.

With regards to heterogeneity, how do you account for the uniqueness of the location from which the biopsy was obtained?

We cannot obtain complete tumors. We address Intratumoral heterogeneity by using multiple tumor models of the same type and many thousands of tumoroids within each study.

Culture and Growth Conditions

Do you use tumor chunks/fragments, or create spheroids of a specific size where there is loss of the original tumor architecture for the recreation of a 3D structure?

Our technology uses mechanically apportioned tumor to create Patient Derived Tumoroids (PDT’s). We do not create tumoroids of a single size but of a variety of sizes from 50-500 μm³. We think of it as growth of the tumor ex-vivo while retaining the tumor architecture and microenvironment.

What is the rationale for using mechanical separation over enzymatic digestion?

We do this to ensure the stromal cells and the source tumor-microenvironment (TME) from the patients remain intact.

Co-Culturing with PBMCs

Are the tumor models tested using autologous PBMC’s?

The Spanios PDT’s use HLA matched allogeneic PBMCs. We do it this way to enable repeatability of each batch for future experiments. Depending on your study goals, we can procure matched live tumor and PBMCs from the same patient if requested.

How do you activate your PBMCs and ensure proliferation?

We add CD-28 and IL-2 to ensure activation. We have demonstrated that B-cells and macrophages are present. Yes, they can proliferate but we don’t allow them to change the TME.

Co-Culturing with Perfusion System

How do you intend to utilize your Perfusion Model to capture the patient response for complex modalities?

We do this with our next generation dynamic PDTs platform COMPASS 3 which utilizes a blood substitute. Both our COMPASS 2 and COMPASS 3 platforms help to better stratify your patients.

What is the benefit of the COMPASS 3 Perfusion Model? Is it primarily to investigate immune incorporation?

The COMPASS 3 Perfusion Model is a more physiologically relevant platform as it works with a blood substitute to test agents in the presence of an oxygenated microenvironment versus hypoxic conditions. This can be done with or without the immune system components being present.

Immune-Competence

Does your platform have stroma and the immune components?

This is dependent on the version of the model systems. COMPASS 1 platform models have the epithelium and stroma; while COMPASS 2 has both stroma and immune components. The COMPASS 3 Platform can model both all of the aforementioned both with and without oxygen modulation.

Are myeloids present in the models?

We have tested B cells, T cells and Macrophages so far. We are in the process of testing all pertinent immunophenotypes including NK cells and Dendritics.

Regarding T-cell engagement, NK cells, can you find signs of engagement and signs of exhaustion?

We can demonstrate this by performing cytokine studies. For example, we can test CD-28, CD-169 interaction through cytokines. We also have done checkpoint inhibitor studies. In immune competent organoids, we test anti-PD1 and anti-CTLA4. We have tested these in multiple tumoroids per sample in several indications over 14 days with great success.

Study Scope and Design

How comparable are Spanios PDT results to clinical outcomes?

Previous studies have shown that 80-85% of what has worked intratumorally has also worked in the clinical setup. Spanios PDT’s are the most translatable model available.

What is your broad framework of studies that you do?

Apart from efficacy studies we focus a lot on mechanistic and biomarkers studies, and other routine ways of helping in patient selection criteria for clinical studies.

How many treatment arms are possible (average) per specimen (it was mentioned that 4 replicates are run per treatment arm)?

We do not have a limit on the treatment arms as our PDTs are always banked thus we can build as many arms as required. We suggest an n=4. We also usually do not have a limit on the number of assays that can be performed.

Readouts

What testing readouts do you offer?

We provide details of tumor volume reduction and tumor number reduction, both bulk and scRNA-seq, immunofluorescence, immunophenotyping, apoptosis, proliferation and migration assays, cytokine arrays, tumor cell and non-tumor cell high content imaging, MOA studies, Escape-response assessments, FACS, deep proteomics and H&E staining. Additional assays available upon request. Fully customizable.

For cell therapies, could cell migration/tumor infiltration be followed and quantified via a cell fluorescence label?

Yes, we have followed the PBMCs by labeling them so TIL infiltration can be followed using a fluorescent label. Cell migration data is usually done using phase contrast microscopy over time.