Fine-Needle Aspiration-Based Patient-Derived Cancer Organoids

Abstract

Patient-derived cancer organoids hold great potential to accurately model and predict therapeutic responses. Efficient organoid isolation methods that minimize post-collection manipulation of tissues would improve adaptability, accuracy, and applicability to both experimental and real-time clinical settings. Here we present a simple and minimally invasive fine-needle aspiration (FNA)-based organoid culture technique using a variety of tumor types including gastrointestinal, thyroid, melanoma, and kidney. This method isolates organoids directly from patients at the bedside or from resected tissues, requiring minimal tissue processing while preserving the histologic growth patterns and infiltrating immune cells. Finally, we illustrate diverse downstream applications of this technique including in vitro high-throughput chemotherapeutic screens, in situ immune cell characterization, and in vivo patient-derived xenografts. Thus, routine clinical FNA-based collection techniques represent an unappreciated substantial source of material that can be exploited to generate tumor organoids from a variety of tumor types for both discovery and clinical applications.

Keywords: Cancer; Clinical Medicine; Tissue Engineering.

Graphical abstract

Figure 1

FNA-Based Patient-Derived Organoid Model (A) Fine-needle aspiration can be performed on patients, surgical specimens, or animals. One to three needle passes are typically collected, and the needle is rinsed in RPMI 1640 or DMEM. Following this rinse, the cells can be directly plated in either a semisolid or disc organoid format. (B) Organoids begin to form within 1 week of plating and have morphologies unique to each individual patient. Three melanoma organoids shown have distinct morphology on 20× bright-field imaging. (C) Organoids can also be embedded in paraffin blocks for morphologic evaluation using H&E staining, images taken at 20× magnification (scale bar, 50 μm). Organoid morphology, as seen on H&E stain, closely recapitulates the primary patient tumor morphology across multiple tumor types including melanoma, gastrointestinal carcinomas, thyroid carcinomas, and renal cell carcinoma.

Figure 2

Patient-Derived Organoids Maintain Identical Patient Morphology and Immunophenotype in Culture (A) Three distinct FNA patient-derived organoids (FNA-PDOs) for melanoma. These organoids also maintain S-100 and Ki-67 immunohistochemical (IHC) staining similar to the patient tumor (20× magnification; scale bar, 50 μm). (B) Two distinct FNA-PDOs for thyroid cancer maintain expression of markers indicative of thyroid differentiation, AE1/AE3 and TTF-1, on IHC (20× magnification; scale bar, 50 μm). (C) Gastrointestinal and renal FNA-PDOs demonstrate preserved AE1/AE3 expression (20× magnification; scale bar, 50 μm). (D) Immunofluorescence staining of the two thyroid cancer FNA-PDOs (shown in B) highlights both a population of tumor cells (green, CK8/18 positive staining) as well as a supportive population of cancer-associated fibroblasts (CAFs, red, SMA; DNA in blue; 20× magnification; scale bar, 100 μm). (E) Disc organoid cultures for numerous tumor types tested demonstrate 2D cultures that extend from organoids and grow along the bottom of the plate. Bright-field images of melanoma FNA-PDO taken at 4 and 6 days of culture showing rapid growth of the 2D population (10× magnification; scale bar, 100 μm). (F) Immunofluorescence of these 2D cultures (thyroid FNA-PDO shown) demonstrates both cancer epithelial cells (CK8/18, green) and cancer-associated fibroblasts (CAFs, SMA, red; 10× magnification; scale bar, 100 μm).

Figure 3

FNA-Based Patient-Derived Organoids are Readily Adapted to High-throughput Screening (A) FNAs directly plated in semisolid organoid culture can be tested for drug sensitivity. Using 24-well plates, BRAF^V600E-mutant (left) and BRAF-wild-type (right) thyroid cancer organoids were treated with dabrafenib, a BRAF inhibitor (30 nM), for 20 days. BRAF^V600E-mutant organoids show decreased organoid size and number following treatment. As expected, BRAF-wild-type organoids show no observable response. Images representative of three replicates. ∗∗p < 0.05, Mann Whitney test. (B) FNA-based patient-derived gastric signet ring organoids were assayed for drug sensitivity using disc-organoid MTT assay in 96-well plates. Images representative of four replicates; error bars represent standard deviation. ∗ = p < 0.05 (Student’s t test). The x axis indicates the concentrations of each drug (alone or in combination); the y axes are MTT signals normalized to the highest absorbance value within each experiment. (C) FNA-PDOs from aggressive anaplastic thyroid cancer were assessed for viability following doxorubicin treatment using an automated high-throughput 384-well assay. The thyroid cancer FNA-PDOs showed response to doxorubicin. Automated high content imaging also confirms organoid growth in the 384-well format, as seen in these untreated melanoma FNA-PDOs. Images representative of three replicates; error bars represent standard error of the mean (SEM). (D) Using a high-throughput fluorescent assay, patient-derived melanoma organoids were labeled with Calcein AM (green fluorescence, labels live cells) and propidium iodide (red fluorescence, labels dead cells). To visualize both live and dead cells, a cell-permeable DNA stain Hoechst 33342 was used. Although the overall organoid structure remained intact, the organoid decreased in size and cells in the organoids were killed following 3 days of treatment with MEK inhibitor, trametinib (0.1 μM), and BRAF inhibitor, dabrafenib (10 μm). This is in agreement with clinical data showing that dabrafenib and trametinib combination is effective for treatment of BRAF V600E melanoma.³¹ Images representative of three replicates; scale bar, 100 μm. (E) Organoids from indicated human melanoma tumors were generated using FNA of corresponding PDX tumors grown in BALB/c nu/nu mice. Organoids were plated in semisolid media into 96-well low-attachment plates and treated with 5 μm AMG-232, 1 μm debrafenib, 0.1 μm trametinib, or combination of three drugs for 3 days. Calcein M/Propidium Iodide viability assay was performed as shown in (C). The ratio of the intensity of red (dead cells) and green (live cells) fluorescent signal in individual organoids was plotted. Each dot represents an individual organoid. We analyzed 9–23 organoids per group for PDO2552, 11–24 for PDO1668, 17–43 for PDO 9164, and 8–14 for PDO 2132. Unpaired t test was used to determine the significance of differences between indicated treatment groups. Error bars represent standard deviation.

Figure 4

FNA-Based Patient-Derived RCC Organoids Contain Immune Cells from the Tumor Microenvironment (A) Immune cells are extracted and plated during FNA-PDO collection and generation. This representative RCC FNA-PDO demonstrates classic morphology by H&E with positive cytokeratin staining (AE1/AE3) and infiltration of B cells (CD20), T cells (CD3), and macrophages (CD163) by immunohistochemistry (IHC). (B) Cell viability at collection was assessed using flow cytometry on fresh FNA material collected from RCCs versus fresh material following tumor digestion of RCCs, showing similar overall viability and CD45+ viability between FNA and digestion extraction methods (n = 4 tumors). Error bars represent standard error of the mean (SEM). (C) Both FNA and digestion methods have similar lymphoid subset viability at collection (n = 4 tumors, error bars represent SEM). (D) Both FNA and digestion methods have similar myeloid subset viability at collection (n = 4 tumors, error bars represent SEM). (E) Absolute immune cell counts at collection for FNA (nine needle passes per tumor) and digestion (1 cm³ tissue per tumor completely digested, n = 4 tumors, error bars represent SEM). (F) RCC FNA-PDO and digestion-PDO immune cell recovery counts at 2 weeks of disc culture demonstrate significantly improved survival of CD45+ cells from FNA-PDOs as compared with digestion-PDOs in both Wnt3A-enriched and non-Wnt-enriched complete media (n = 4 tumors, error bars represent SEM, ∗p < 0.05). (G) FNA-PDO CD45+ cell recovery counts at 2 weeks in culture demonstrates improved CD45+ cell survival with both Wnt-enriched media and 100 IU/mL IL-2 supplementation (n = 2 tumors, error bars represent SEM, ∗p < 0.05). (H) FNA-PDO lymphocyte recovery counts at 2 weeks in culture demonstrates CD3+ lymphocyte survival in all conditions and improved CD8+ lymphocyte survival with Wnt-enriched media and IL-2 supplementation (n = 2 tumors, error bars represent SEM, ∗p < 0.05). (I) FNA-PDO myeloid recovery counts at 2 weeks in culture demonstrates improved survival of CD14+CD11b+ myeloid cells when cultured with Wnt-enriched media (n = 2 tumors, error bars represent SEM, ∗p=<0.05).