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Abstract
Background Transforming preclinical screening of immunotherapies to reveal new immunotherapeutic modalities requires model platform that gives insight into the infiltration of immune components to provide spatial and temporal information on the immunosuppressive and immunomodulatory mechanisms at play. One significant hurdle in building tumoroids that can address this need is to correctly incorporate cellular complexity in a controlled manner. We are conducting investigative studies on the infiltration depth, extent, and time of T Cells (CD3+), B cells (CD20), Macrophages (CD68), and NK cells (CD16) in two different cancer indications to enable multiple forms of therapeutic models that can be tested on our model platform. We used Immunofluorescences (IF) for the identification and quantification of the subsets that are integrated into PBMC-stimulated organoids.
Methods The PDT culture (S-PDT-V1) of Carcinosarcoma and gall bladder cancer (GBC) from Spanios tumoroid biorepository was cultured, recovered from the Matrigel based matrix, and seeded in 24 well plates. The PDTs were then harvested, fixed, and washed before blocking them for antibody treatment. Primary antibodies for CD3, CD20, CD16, and CD68 were used followed by secondary antibody and DAPI staining. Prior to evaluating them over time, the PBMCs for the four immune markers were tested on Day 0 of growth using immunocytochemistry and imaged using Leica inverted fluorescent scope (40x). The PDT growth using the same PBMCs was then evaluated for Days 3 and 7 (figure 1).
Results Our initial test of PBMCs for immune markers CD3, CD20, CD68, and CD16 showed presence of makers on Day 0 of growth (figure 2). Using the same PBMC at later time points clearly showed the presence of the immune markers on Day 3 (figure 3) and Day 7 (figure 4) post-growth as well. Similar results were observed in both the Sarcoma and GBC PDTs (figures 5 and 6).
Conclusions The results demonstrate that our model platform with intact immune components including T cells, B cells, macrophages, and NK cells has the potential to bridge the gap by recreation of TME features as close as possible to in vivo conditions. We are also evaluating the tumoroid infiltration of other immune components and immunophenotypes using flow cytometry; analyze these immune components individually and together using spatial RNA seq; and evaluate the impact of oxygenation. We will be testing for the functionality of the immune components in the context of immunotherapy in the future.
Schematic workflow diagram. Immunofluorescence testing of immune components in Carcinosarcoma and Gall Bladder Carcinoma (GBC) patient derived tumoroids on days 3 & 7.
Testing PBMC for availability of the immune components. The PBMCs used for the study were tested for presence of immune components of interest. This included the T cells, B cells, NK and Macrophages. A monolayer or 2D of PBMC was cultured for 24hr after which IF was performed to determine the presence of specific immune components. The images were taken using Leica inverted fluorescent scope (20x) on Day 0.
Testing for presence of immune components in immune competent Carcinosarcoma tumoroids on Day 3. High-grade Carcinosarcoma were cultured from Spanios tumoroid Biorepository. After the third passage, the tumoroids were recovered harvested, fixed and washed before blocking them for antibody treatment. Primary antibody were for CD3, CD20, CD16 & CD68 followed by secondary anti body and DAPI staining. The slides mounted, and images were taken using Leica inverted fluorescent scope (40×) on Day 3.
Testing for presence of immune components in immune competent Carcinosarcoma tumoroids on Day 7. High-grade Carcinosarcoma were cultured from Spanios tumoroid Biorepository. After the third passage, the tumoroids were recovered harvested, fixed and washed before blocking them for antibody treatment. Primary antibody were for CD3, CD20, CD16 & CD68 followed by secondary antibody and DAPI staining. The slides were mounted, and images were taken using Leica inverted fluorescent scope (40×) on Day 7.
Testing for presence of immune components in immune competent Call Bladder Carcinoma tumoroids on Day 3, Moderately Differentiated GBC were cultured from Spanios tumoroid Biorepository. After the third passage, the tumoroids were recovered harvested, fixed and washed before blocking them for antibody treatment. Primary antibody were for CD3, CD20, CD16 & CD68 followed by secondary antibody and DAPI staining. The slides were mounted, and images were taken using Leica inverted fluorescent scope (40×) on Day 3.
Testing for presence of immune components in immune competent Call Bladder Carcinoma tumoroids on Day 7. Moderately Differentiated GBC were cultured from Spanios tumoroid Biorepository. After the third passage, the tumoroids were recovered harvested, fixed and washed before blocking them for antibody treatment. Primary antibody were for CD3, CD20, CD16 & CD68 followed by secondary antibody and DAPI staining. The slides were mounted, and images were taken using Leica inverted fl uorescent scope (40×) on Day 7.
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