Application of 3D tumoroid systems to define immune and cytotoxic therapeutic responses based on tumoroid and tissue slice culture molecular signatures

Abstract

We have developed 3D-tumoroids and tumor slice in vitro culture systems from surgical tumor specimens derived from patients with colorectal cancer (CRC) or lung cancer to evaluate immune cell populations infiltrating cultured tissues. The system incorporates patient's peripherally and tumor-derived immune cells into tumoroid in vitro cultures to evaluate the ability of the culture to mimic an immunosuppressive tumor microenvironment (ITM). This system enables analysis of tumor response to standard therapy within weeks of surgical resection. Here we show that tumoroid cultures from a CRC patient are highly sensitive to the thymidylate synthase inhibitor 5-fluorouracil (adrucil) but less sensitive to the combination of nucleoside analog trifluridine and thymidine phosphorylase inhibitor tipiracil (Lonsurf). Moreover, re-introduction of isolated immune cells derived from surrounding and infiltrating tumor tissue as well as CD45+ tumor infiltrating hematopoietic cells displayed prolonged (>10 days) survival in co-culture. Established tumor slice cultures were found to contain both an outer epithelial and inner stromal cell compartment mimicking tumor structure in vivo. Collectively, these data suggest that, 3D-tumoroid and slice culture assays may provide a feasible in vitro approach to assess efficacy of novel therapeutics in the context of heterogeneous tumor-associated cell types including immune and non-transformed stromal cells. In addition, delineating the impact of therapeutics on immune cells, and cell types involved in therapeutic resistance mechanisms may be possible in general or for patient-specific responses.

Keywords: 3D; biomarkers; colorectal cancer; immune cells; organoid.

Figure 1. 3D colon cancer organoid cultures with growth in vitro and expression of tumor-associated markers

A. Bright field imaging of 3D organoid cultures of normal colon (N) and colorectal cancer (CRC) (T). B. Calcein uptake in CRC tumoroid culture subjected to selection using growth factor deprivation. Immunofluorescence for tumor markers p53, EpCam and beta-catenin C., D.

Figure 2. 3D air-liquid interface (ALI) lung cancer in vitro cultures express several tumor serum markers in culture over extended culture periods

A., B. IHC for CA19-9 and CEA expression in squamous cell carcinoma and CRC tumor-of-origin and in ALI tumor cultures. C., D. Assessment of CEA and CA19-9 in patient serum and 3D culture media over time. Squamous cell carcinoma (SCC) ALI. T1-44d - days in culture following isolation. Δ29d - 29 days before sampling.

Figure 3. Immune profiling of patient donor tissues and the expression of immune cell markers from 3D ALI cultures in vitro

IHC for CD45+ and CD3+ cells in a CRC and the corresponding 3D organoid at eight (8) days after initial culture.

Figure 4. Assessment of CRC patient tumoroid responses to cytotoxic drugs in vitro at 2 weeks following tumor resection

. A. Bright field imaging of 3D organoid cultures from a colorectal cancer (CRC). B. CellTiter-Glo (ATP content) was used to determine the dose-response characteristics of a CRC tumoroid following treatment with FDA-approved drugs and trametinib. A duplicate 12-point dose titer was used for each drug that was investigated with 5-10 organoids per 96-well. C. H/E staining of the CRC of origin and tumor organoid cultures. D. Dose-response relationship of FDA- approved drugs and trametinib in CRC tumoroid cultures.