Establishment of two novel organoid lines from patients with combined hepatocellular cholangiocarcinoma

doi:10.21203/rs.3.rs-4935983/v1

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Establishment of two novel organoid lines from patients with combined hepatocellular cholangiocarcinoma

https://doi.org/10.21203/rs.3.rs-4935983/v1

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Combined hepatocellular cholangiocarcinoma (cHCC-CCA) is a unique subtype of primary liver cancer displaying both hepatocytic and cholangiocytic differentiation. The development of effective treatments for cHCC-CCA remains challenging because of its high heterogeneity and lack of a suitable model system. Using a three-dimensional culture system, we successfully established two novel cHCC-CCA organoid lines from patients undergoing surgical resection for primary liver cancer. cHCC-CCA organoid lines were authenticated by fingerprint analysis, and their morphology, growth kinetics, and anchorage-independent growth were also characterized. Hematoxylin and eosin staining and immunohistochemical analysis showed that the cHCC-CCA organoids preserved the growth pattern, differentiation grade, and phenotypic characteristics of their parental tumors. Whole-exome sequencing demonstrated that patient-derived cHCC-CCA organoid lines retained the genetic alterations identified in their original tumors. Subcutaneous tumors developed in immunodeficient mice after injection of cHCC-CCA organoids. Histologically, the xenografts recapitulated the features of the original cHCC-CCA tumors, harboring both HCC and intrahepatic cholangiocarcinoma components within the same tumor. The establishment of patient-derived cHCC-CCA organoid lines with high tumorigenicity provides a valuable resource for the mechanistic investigation and drug development of this disease.

combined hepatocellular cholangiocarcinoma

patient-derived organoids

hepatocytic differentiation

cholangiocytic differentiation

tumorigenicity

Combined hepatocellular cholangiocarcinoma (cHCC-CCA) is a rare primary liver cancer (PLC) showing histological features of both HCC and intrahepatic cholangiocarcinoma (ICC). Its incidence ranges from 0.4–14.2% of all PLCs [1, 2].The terminology and diagnostic criteria of cHCC-CCA has continuously evolved since it was first reported by Gideon Wells in 1903 [3]. The fifth edition of WHO classification published in 2019 defines cHCC-CCA as a tumor entity that contains unequivocal elements of both HCC and ICC within the same tumor morphologically, using hematoxylin and eosin (H&E) staining. In general, the prognosis of cHCC-CCA is poorer than that of HCC and closer to that of ICC. Currently, the management of cHCC-CCA is not yet standardized, and surgical resection with lymph node dissection remains the only curative option for this condition. However, the 5-year recurrence rate after surgical resection is as high as 80%, and the 5-year survival rate is not more than 30% [4]. Neither sorafenib (the first-line treatment for advanced HCC) nor the combination of gemcitabine and cisplatin (the standard of care for unresectable ICC) has yielded significant clinical benefits in cHCC-CCA treatment [2, 5]. Therefore, gaining an in-depth understanding of these phenotypically heterogeneous tumors and developing novel therapeutic strategies for patients with cHCC-CCA are urgently needed.

Despite tremendous improvements in primary culturing of human cancer cells, it remains very difficult to establish high-quality, permanent cell lines from PLC tumors, especially for cHCC-CCA. Thus far, only two two-dimensional (2D) cHCC-CCA cell lines have been established [6, 7]. In addition, cancer cells grown in 2D monolayer culture are morphologically flattened and enlarged and cannot retain the histological and genetic characteristics of their parental tumors, which affects their responses to therapeutic agents. Patient-derived organoids (PDOs) are recently developed cancer models with potential for application in drug screening and personalized treatments due to their ability to preserve the features of the original tumors [8–10].To increase the number and diversity of clinically relevant models available for cHCC-CCA study, we successfully established two organoid lines from patients undergoing surgical resection for PLC.

Isolation and culture of human cHCC-CCA organoid lines

cHCC-CCA tumor and paired adjacent non-tumor liver tissues were obtained from patients undergoing surgical resection for PLC at the First Affiliated Hospital of Nanjing Medical University. This study was approved by the Ethics Committee of Nanjing Medical University, and the patients provided written informed consents. The isolation and culture of human cHCC-CCA and normal cholangiocyte organoids were performed as described previously, with minor modifications [11]. Briefly, tissues were minced under sterile conditions, digested with liver digestion medium (Thermo Fisher Scientific, Waltham, MA, USA) at 37 ℃ for 30–60 min, and centrifuged at 200 × g for 5 min. After washing twice with cold Advanced DMEM/F12 (Gibco, Grand Island, NY, USA), the pellet was mixed with Type 2 Basement Membrane Extract (BME2; R&D, Minneapolis, MN, USA), and seeded into ultra-low attachment 24-well plates (Corning Inc., Corning, NY, USA) (2,000–5,000 cells/well). Following solidification of BME2, 600 µL of culture medium was added to each well. The cHCC-CCA organoid culture medium was based on advanced DMEM/F12 medium supplemented with 30% (vol/vol) Wnt-3A conditioned medium (CM), 10% (vol/vol) Rspo-1 CM, 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA), 1% Glutamax (Invitrogen), 10 mM HEPES (Sigma-Aldrich, St. Louis, MO, USA), 1:50 B27 (Thermo Fisher Scientific), 1:100 N2 supplement (Thermo Fisher Scientific), 2.0 µM A8301 (R&D), 1.25 mM N-acetyl-l-cysteine (Sigma-Aldrich), 10 µM forskolin (R&D), 10 mM nicotinamide (Merck KGaA, Darmstadt, Germany), 10 µM rock inhibitor Y27632 (R&D), 10 nM recombinant human (Leu15)-gastrin I (Thermo Fisher Scientific), 50 ng/mL recombinant human EGF (R&D), 100 ng/mL recombinant human FGF10 (R&D), 25 ng/mL recombinant human HGF (R&D), and 50 ng/mL recombinant human noggin (R&D). After initial culturing for 10–14 days, organoids were dissociated with dispase for 15 min, followed by TrypLE Express (Thermo Fisher Scientific) digestion for 3–5 min. Organoids were then passaged at a ratio of 1:4–1:6 every 7–9 days.

Fingerprint analysis

Genomic DNA was extracted from cHCC-CCA organoids and their corresponding tumor tissues using the QIAamp DNA Mini Kit (Qiagen, Duesseldorf, Germany). Then, the genomic DNA was subjected to fingerprint analysis, which was conducted by Cellcook Biotech Co., Ltd. (Guangzhou, China). A total of 23 short tandem repeats (STR) loci were amplified in each sample, and alleles of each locus were determined using GeneMapper ID Software (Thermo Fisher Scientific).

Histology and immunohistochemistry (IHC) analysis

Tissues and organoids were fixed in 4% paraformaldehyde, dehydrated, paraffin-embedded, and sectioned. The sections were subjected to H&E staining, and blindly evaluated by two experienced pathologists who specialized in hepatopathology. IHC analysis was conducted as described previously [12] using primary antibodies targeting alpha fetoprotein (AFP) (ab46799, 1:250, Abcam, Boston, MA, USA), Glypican 3 (GPC3, ab66596, 1:100, Abcam), CPS1 (ab129076, 1:250, Abcam), Cytokeratin 19 (KRT19, ab76539, 1:1000, Abcam), MUC1 (ab109185, 1:250, Abcam), and Nestin (ab176571, 1:1000, Abcam). The sections were incubated with primary antibodies overnight at 4 ℃. The sections were rinsed with washing buffer and incubated with goat anti-rabbit IgG secondary antibody (ab97080, 1:1,000, Abcam) for 1 h. Reactivity was detected using a Dako EnVision Detection System (Dako, Hamburg, Germany) following manufacturer’s instructions.

Whole-exome sequencing (WES)

Genomic DNA was extracted from cHCC-CCA organoids, corresponding tumor tissues, and adjacent non-tumor liver tissues using the Hipure Universal DNA kit (Magen, Guangzhou, China). The DNA libraries were prepared by shearing the samples, followed by end repair, phosphorylation, and ligation to barcoded sequencing adaptors. Ligated DNA fragments with lengths between 200 and 350 bp were selected and enriched using the SureSelect Human All Exon V6 Kit (Agilent Technologies, Palo Alto, CA, USA). Sequencing was conducted using the Illumina NovaSeq 6000 (San Diego, CA, USA). The reads were aligned to the GRCh38 reference using the Burrows-Wheeler Aligner, and variant calling was performed using the Genome Analysis Toolkit (GATK) Unified Genotyper with local realignment and base quality score recalibration. SNPs and InDels were filtered using GATK's Variant Filtration with appropriate standards, and variants exhibiting segregation distortion or sequencing errors were discarded. Cancer-related variants were determined by performing the following filtering steps: (i) filtering out variants with reads supporting variations present in adjacent non-tumor liver tissues, (ii) filtering out variants present in dbSNP, (iii) filtering out variants with a frequency of > 0.01 in the ExAC database (v03), (iv) filtering out synonymous and intronic variants, and (v) filtering out variants with SIFT scores > 0.05.

Growth kinetics of cHCC-CCA organoid lines

After digestion with dispase and TrypLE Express as described above, cHCC-CCA organoids were mixed with BME2 and seeded into ultra-low attachment 24-well plates at 20,000 cells/well. Organoids were harvested, dissociated into single cells, and counted every day for 8 consecutive days, and the counts were performed in triplicate. Doubling times were calculated from cHCC-CCA organoids during their exponential growth phase using the following formula:

DT = Δt × [ln2/(lnNt − lnN0)], where Δt is the duration of culture, Nt is the number of cells at the end of incubation, and N0 is the cell number at the beginning of incubation.

Soft agar colony formation assay

To evaluate the anchorage-independent growth of cHCC-CCA organoids, the soft agar colony formation assay was performed. Briefly, organoids were harvested and dissociated into single cells, suspended in 0.5 mL 0.4% agar, and layered over 0.8% agar in 24-well plates at a density of 2,000 cells/well. After incubation at 37 ℃ for 10 days, cells were stained with 1 mg/mL iodonitrotetrazolium chloride (Sigma-Aldrich). All experiments were performed in triplicate.

Lentiviral transduction of cHCC-CCA organoids

cHCC-CCA organoids were harvested and digested into single cells (approximately 2×10⁵), resuspended in culture medium containing 10⁷ EU GMLV-CMV-MCS-ZsGreen1-T2A-Luc-P2A-Puro lentivirus (Jiman Biotechnology, Shanghai, China) and 6 µg/mL polybrene (Sigma, St. Louis, MO, USA), and plated into ultra-low attachment 24-well plates coated with BME2 20 min before the experiment. After 24 h of incubation, the virus-containing medium was replaced with fresh culture medium. Infection efficiency was monitored by observing the number of cells expressing green fluorescent protein (GFP) under a fluorescent microscope.

In vivo tumorigenic assay of cHCC-CCA organoids

Male immunodeficient NCG mice (4–6 weeks old, GemPharmatech Co., Ltd., Nanjing, China) were bred at the Animal Core Facility of Nanjing Medical University and kept under specific pathogen-free conditions. All animal procedures were approved by the Animal Experimentation Ethics Committee of Nanjing Medical University. cHCC-CCA organoids were collected and released from BME2 by digestion with dispase for 15 min. Approximately 1 × 10⁶ organoid cells were suspended in 100 µL BME3/PBS mixture (1:2) and subcutaneously implanted into the left flank of each mouse. Tumor size was measured by a caliper every week and luciferase signals were detected using IVIS Lumina Series III (PerkinElmer, Waltham, MA, USA) following intraperitoneal injection of 150 mg/kg D-Luciferin (Cayman Chemical, Ann Arbor, MI, USA). When the xenografts reached approximately 1 cm in diameter, mice were sacrificed. The tumors were removed, fixed in 10% formalin, and embedded in paraffin.

Establishment of patient-derived cHCC-CCA organoid lines

We established two cHCC-CCA organoid lines using tumor tissues from untreated patients undergoing surgical resection for PLC. The clinical features of the patients are shown in Table 1. The two organoid lines were designated as T63_ORG and T142_ORG respectively. Morphogically, the majority of T63_ORG organoids grew as solid/compact spheroids, although a small number formed glandular lumen. T142_ORG organoids presented as homogeneous compact spheroids with pseudoglandular structures. Generally, the size of T142_ORG organoids was smaller than that of T63_ORG organoids. In contrast with the cHCC-CCA organoids, the normal cholangiocyte organoids (Chol63_ORG and Chol142_ORG) derived from adjacent non-tumor liver tissues always presented as single-cell layered cyst-like hollow structures (Fig. 1A). The two cHCC-CCA organoid lines were stably passaged for over 50 generations (> 1 year), and showed no morphological signs of apoptosis or senescence (Supplementary Fig. 1).

Table 1

Clinical data of patients and characteristics of tumors
Patient	Sex	Age	Tumor	Etiology	Edmondson	Cirrhosis	AFP (IU/mL)
63	Male	49	cHCC-CCA	HBV	III	Yes	176.5
142	Male	64	cHCC-CCA	HBV	II	Yes	31.09
AFP, alpha-fetoprotein; HBV, hepatitis B virus; cHCC-CCA, combined hepatocellular cholangiocarcinoma.

The growth curve of T63_ORG and T142_ORG at the eighth passage displayed a stationary phase at the early and final stage of the experiment (Fig. 1B). After 3 days of slow proliferation, the organoids entered the rapid logarithmic growth phases that lasted for 2–3 days and plateaued on days 6–7. The expansion potential of T63_ORG organoids is greater than that of T142_ORG organoids. The doubling times of T63_ORG and T142_ORG organoids were 26 h and 32 h respectively. Anchorage-independent growth ability of cHCC-CCA organoids was evaluated by soft agar colony formation assay. Both T63_ORG and T142_ORG organoids formed colonies in soft agar, whereas the two normal cholangiocyte organoid lines did not, validating the malignant phenotype of the two cHCC-CCA organoid lines (Fig. 1C).

Authentication of cHCC-CCA organoid lines

We authenticated cHCC-CCA organoid lines by conducting STR analysis and found that T63_ORG and T142_ORG organoids closely matched with their parental tumors (Supplementary Fig. 2), and did not align with any of the cell lines deposited in public cell banks. Also, mycoplasma testing was negative for both cHCC-CCA organoid lines (data not shown).

Genetic characteristics of cHCC-CCA organoid lines

WES was conducted to assess the retention of the mutation landscape of the parental tumors in the cHCC-CCA organoids. There were 57 and 73 cancer-related mutations respectively in the original tumors from patient 63 and 142, which was consistent with published mutation burdens for cHCC-CCA [15]. These mutations were well preserved in the corresponding cHCC-CCA organoid lines (supplementary table 1–4), with 84.2% retained in T63_ORG organoids and 91.8% retained in T142_ORG organoids. Some mutations in cHCC-CCA organoid lines were not identified in the original tumors (Fig. 1D). It is unlikely that these specific mutations were generated during the organoid culture process because the two organoid lines collected for WES were all at their early passage (4th passage). We speculate that these specific mutations may be enriched in the cHCC-CCA organoids but were masked by various cell populations in the original tumor tissues, such as stromal, inflammatory, and necrotic cells. This phenomenon has been previously described during organoid culture [11, 16]. Mutations in TP53, one of the most mutated genes in cHCC-CCA [15, 17], were identified in both organoid lines and their original tumors. Additionally, some recurrently mutated genes in HCC including AR, KEAP1, MUC16, NTRK2, and TRIM3 [11, 18–20], were found in the two tumor-organoid pairs (Fig. 1E). However, other commonly mutated genes in ICC, such as KRAS, IDH1/2, ARID1A, and CDKN2A/B [21, 22], were not mutated in either tumor-organoid pair. The genetic alterations in the two cHCC-CCA organoid lines and their parental tumors revealed that they had similar genetics to HCC, which was consistent with the previously reported genomic profiling of cHCC-CCA [23]. These results demonstrated that our established cHCC-CCA organoid lines recapitulated the genetic signature of their original tumors.

Histopathological features of cHCC-CCA organoid lines

Histologically, the two original tumors showed typical characteristics of cHCC-CCA, which consists of both HCC and ICC components within the same tumor. In the HCC component, tumor cells displayed a trabecular growth pattern, whereas the ICC component was characterized by glandular structures embedded in abundant fibrous stroma (Fig. 2A). cHCC-CCA organoids retained the growth pattern and differentiation grade of the parental tumors. The majority of T63_ORG organoids presented as solid spheroids, and a small number formed glandular lumen-like structures. T142_ORG organoids showed a solid growth pattern and formed pseudoglandular rosettes, a feature which was also observed in the HCC component of the original tumor from patient 142. In addition, T63_ORG and T142_ORG organoids exhibited epithelial-like features with a high nucleus to cytoplasm ratio and were classified as Edmondson differentiation grade III and II respectively, which was consistent with their corresponding tumors (Fig. 2B).

IHC analysis showed that HCC differentiation markers AFP, GPC3 and Hep Par 1 were weakly to moderately expressed in T63_ORG and T142_ORG organoids, displaying similar distribution and expression intensity as their original tumors. The same was true for cholangiocytic differentiation markers KRT19 and MUC1, which were strongly expressed in T63_ORG and T142_ORG organoids, as observed in their parental tumors. Nestin, an embryonic type VI intermediate filament protein, was recently reported to be overexpressed in most of cHCC-CCA tumors and may be a potential biomarker for diagnosis of cHCC-CCA [13, 14]. In this study, both cHCC-CCA organoid lines and their original tumors exhibited strong expression of this protein, further confirming their entity as cHCC-CCA (Fig. 3).

Tumorigenicity of cHCC-CCA organoid lines in vivo

To evaluate the tumorigenic potential of cHCC-CCA organoids, T63_ORG and T142_ORG organoids were transduced with lentivirus containing luciferase and GFP dual reporter genes. Both T63_ORG and T142_ORG organoids were transduced efficiently (Fig. 4A), suggesting that these cHCC-CCA organoids can be easily manipulated experimentally. The cHCC-CCA organoids were then subcutaneously inoculated into immunodeficient NCG mice (n = 3). Approximately 1 month after organoids inoculation, a visible tumor developed, yielding a tumor formation success rate of 100%. The tumors reached a size of around 1 cm in diameter 2 months after inoculation with T63_ORG organoids and 3 months after inoculation with T142_ORG organoids (Fig. 4B). Anatomical dissections of these mice revealed no metastatic lesions in the liver or lungs, indicating a non-metastatic phenotype for the two xenografts. In accordance with this, no intrahepatic or distant metastasis was found in the two corresponding patients. H&E staining revealed that the xenografts exhibited both hepatocytic and cholangiocytic differentiation (Fig. 4C). The ICC component formed typical glandular lumina within the stroma, whereas HCC component was characterized by trabecular architectures and pseudoglandular rosettes. IHC analysis unveiled the positive expression of MUC1 and Nestin in xenografts, consistent with the results observed in the two cHCC-CCA organoid lines and their corresponding tumors from patients (Fig. 4D).

Currently, therapeutic options for patients with cHCC-CCA are limited, largely due to its high heterogeneity and the lack of relevant preclinical models. In this study, we successfully established two organoid lines representative of cHCC-CCA using surgically resected tumor tissues. STR profiling confirmed that the cHCC-CCA organoid lines were derived from their corresponding tumor tissues and were not contaminated with any of the cell lines listed in public cell banks. H&E and IHC staining indicated that cHCC-CCA organoids retained the growth pattern, differentiation grade, and phenotypic characteristics of their original tumors. WES analysis demonstrated that cHCC-CCA organoids retained genetic alterations present in the parental tumors. The subcutaneous inoculation of T63_ORG and T142_ORG organoids into NCG mice resulted in a 100% formation rate of xenografted tumors, validating the high tumorigenicity of cHCC-CCA organoids in vivo. Most importantly, the PDO-based xenografts (PDOXs) displayed notable histological similarities with their original tumors, which were characterized by co-existence of both HCC and ICC components within the same tumor. Moreover, IHC profiling revealed positive expression of Nestin and MUC1 across the primary tumors, cHCC-CCA organoids, and subcutaneous xenografts, which further substantiated the categorization of T63_ORG and T142_ORG organoids as cHCC-CCA. In 2018, two patient-derived cHCC-CCA organoid lines were first established by Broutier et al [11]. However, they did not assess whether the lines were tumorigenic in vivo. Although Xian et al. claimed that they established several cHCC-CCA organoid lines with tumorigenicity, they did not analyze the STR profiles, genetic alterations and histopathological features of these organoids [24]. Therefore, the identity of these organoids still needs to be validated. To the best of our knowledge, this is the first study to report that the patient-derived cHCC-CCA organoid lines were established, authenticated and fully characterized, and that the features of the original cHCC-CCA tumors were well recapitulated in the PDOXs.

Models of PDOs retain cell–cell and cell–matrix interactions, exhibit a more clinically relevant responses to therapeutic agents than traditional 2D cell lines, improving the success probability for drug screening. However, PDO models lack immune cells and stromal components, limiting their use in the field of immunotherapy. To address this, tumor organoids can be co-cultured with immune cells. For example, Zou et al. co-cultured HCC organoids with T cells and demonstrated that CD39⁺ CAR-T cells effectively killed HBVs⁺ HCC cancer cells using this co-culture system [25]. In recent years, combination use of molecular targeted drugs with immune checkpoint inhibitors (ICIs) has revolutionized HCC treatment, and proved to be effective in ICC treatment. Thus far, very little research has been conducted to evaluate the efficacy of ICIs in the treatment of cHCC-CCA [26]. Therefore, to establish co-culture system of cHCC-CCA organoids with immune cells and to explore the therapeutic potential of ICIs in the treatment of cHCC-CCA will be our next research direction.

In summary, we successfully established two novel patient-derived cHCC-CCA organoid lines with high tumorigenicity, which may advance the understanding of the molecular mechanisms underlying cHCC-CCA and the development of innovative treatments for this disease.

AFP

alpha fetoprotein

BME

basement membrane extract

cHCC-CCA

combined hepatocellular cholangiocarcinoma

conditioned medium

two-dimensional

GFP

green fluorescent protein

GPC3

Glypican 3

ICC

intrahepatic cholangiocarcinoma

ICIs

immune checkpoint inhibitors

IHC

immunohistochemistry

KRT19

Cytokeratin 19

PDO

patients-derived organoids

PDOX

PDO-based xenograft

PLC

primary liver cancer

STR

short tandem repeats

WES

Whole-exome sequencing

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Establishment of two novel organoid lines from patients with combined hepatocellular cholangiocarcinoma

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Abstract

Figures

Introduction

Methods

Isolation and culture of human cHCC-CCA organoid lines

Fingerprint analysis

Histology and immunohistochemistry (IHC) analysis

Whole-exome sequencing (WES)

Growth kinetics of cHCC-CCA organoid lines

Soft agar colony formation assay

Lentiviral transduction of cHCC-CCA organoids

In vivo tumorigenic assay of cHCC-CCA organoids

Results

Establishment of patient-derived cHCC-CCA organoid lines

Authentication of cHCC-CCA organoid lines

Genetic characteristics of cHCC-CCA organoid lines

Histopathological features of cHCC-CCA organoid lines

Tumorigenicity of cHCC-CCA organoid lines in vivo

Discussion

Abbreviations

References

Supplementary Files

Status:

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