2.1 Human subjects

The protocol was reviewed and approved by Memorial Sloan Kettering Cancer Center Institutional Review Board, and by the Rockefeller University Institutional Review Board.

2.2 Biopsies

Adult patients of both genders and whose liver lesions were clinically highly suspicious for HCC, required a diagnostic biopsy were included in the study and provided written informed consent for use of tissue samples. Eligible HCC etiologies were HCV infection as determined by HCV serology and/or detectable HCV RT-PCR; HBV infection as determined by HBV core antibody and/or surface antigen serology, and/or detectable HBV PCR; alcoholic liver disease (ALD) as determined by the history of chronic alcohol use; NAFLD as determined clinically by history, past or current fatty liver on imaging, previous biopsy confirmation and associated metabolic conditions. Biopsies were performed at the interventional radiology department at MSKCC, tumor samples were obtained by image-guidance technique from primary HCC or metastatic site. Biopsy material was cut into small pieces (~0.5 mm³) for implantation.

2.3 Animals

Balb/c Rag2^−/−, NOD Rag1^−/− Il2rg^null (NRG) and Foxn1^nu mice were obtained from Jackson Labs (Bar Harbor, Maine). Mice with a targeted disruption in fumaryl acetoacetate hydrolase (Fah^−/−) were kindly provided by M. Grompe (Oregon Health & Science University) and crossed with Rag2^−/− Il2rg^null or with NRG mice.³¹ Immunodeficient Fah^−/− were bred and maintained on nitisinone (Yecuris, Tualatin, OR) to prevent liver damage. To induce chronic liver injury immunodeficient Fah^−/− mice were cycled off nitisinone after HCC biopsy implantation or kept on continuous nitisinone as non-liver injury controls. At the time of surgery mice were 6–10 weeks of age with weight varying from 20 g up to 30 g. All procedures were reviewed and approved by Rockefeller University Institutional Review Board and Committee on Use and Care of Animals under protocol number 18063.

2.4 Implantation and engraftment monitoring

For intrahepatic implantation, the upper abdomen of mice was shaved and sterilized with iodine. After mice had been anesthetized using isoflurane, a 10–15 mm skin incision was made over the left subcostal margin after which the peritoneum was mobilized. Using a cautery device, 10–15 mm of peritoneum was opened, and the large lobe of the liver was exposed. An ~1 mm opening was created in the center of the lobe and two to three ~0.5 mm³ pieces of biopsy material were loaded into the liver. The outflow tract was cauterized to prevent bleeding and then the peritoneum closed with a Vicryl suture and the skin with hemostat wound clips.

For subrenal capsule (SRC) implantation the left flank of mice was shaved and sterilized with iodine. After mice were anesthetized using isoflurane, a 5–7 mm skin incision was made in the left midaxillary plane, after which the peritoneum was mobilized. Using a cautery device 7–8 mm of peritoneum was opened, the left kidney exposed and at the caudal pole. A ~1 mm opening in the capsule was created. Two to three ~0.5 mm³ pieces of biopsy material were placed under the kidney capsule and moved cranially. The kidney was mobilized back into the peritoneum, closed with a Vicryl suture and the skin approximated with hemostat wound clips.

Engraftment growth was monitored by quantification of human HCC markers in mouse serum every 3–4 weeks by using commercially available human-specific ELISA antibodies and protocols as described previously.³¹ If animal health were adversely affected by tumor growth as shown by displaying lethargy, hunched appearance, ruffled fur, and/or cachexia, the experiment was terminated in accordance with the Rockefeller University Institutional Animal Care and Use Committee (IACUC) protocol.

2.5 Tissue harvest, passaging, and cryopreservation

Once tumors plateaued as assessed by serum markers or imaging, PDX tissues were harvested for histology and cryopreservation. After CO₂ asphyxiation the liver and the left kidney were harvested. Tumors were weighed after separation from the liver or kidney. For tumors in which the kidney could not be separated or identified the median left kidney weight for that strain was subtracted from total weight. Tumor tissue was collected in 10% buffered formalin for histology. The remaining tissue was cut into small, 1–2 mm³ pieces for immediate passaging or put in hepatocyte freezing medium containing 10% DMSO for long-term storage at −150ºC.

2.6 Histological comparison

Samples were formalin-fixed, paraffin-embedded, cut into 5.0 micron sections, and stained with hematoxylin and eosin (H&E). Histopathology was examined under light microscopy.

2.7 Statistical analysis

Pearson correlation coefficient was used to assess the linear correlation between tumor weight of mice with serum hAAT among PDX1. A linear mixed model was used to further evaluate the association between tumor weight and serum hAAT from different PDX. At the biopsy level, Fisher's exact test was used to examine the association between immunodeficiency status and implantation site on the success of PDX formation. To account the clustering of repeated binary observations from mice within a PDX, the generalized estimating equations (GEE) logistic regression model was used to examine the association between the factors mentioned above and PDX formation in the mouse level. To look at the association between liver injured and no liver injury on the 18 biopsies implanted under both conditions, the exact McNemar's test was used. Biopsies that were implanted into Rag2-/- mice were excluded.

All analyses were carried out in GraphPad Prism Software and SAS 9.4 (SAS Institution, Cary, NC). All p-values were two-sided and p-value of 0.05 indicated statistical significance.

3.1 Clinical HCC characteristics and overall PDX formation rate

Sixty-two biopsies were obtained from 60 patients with presumed HCC enrolled in the study. Seventeen samples were excluded from the analysis as they were determined either as cirrhotic liver without HCC (n = 4), a non-HCC cancer (n = 5) or because there was no or not enough tissue for implantation (n = 8). Of the 45 HCC biopsy specimens that were implanted, 37 were obtained from male patients (82%). Patient demographics and tumor characteristics are summarized in Table 1. Our biopsy material selection highlights the HCC etiological distribution most commonly noted in the United States. The underlying liver disease etiologies of the 45 HCC biopsies were HCV (n = 15), alcoholic liver disease (ALD) (n = 6), HBV (n = 8), NAFLD (n = 5), mixed etiology (alcohol and HCV n = 2; alcohol and NAFLD n = 1; HCV and NAFLD n = 1), and no identifiable liver disease etiology or cryptogenic cirrhosis (n = 7).

We found that 20 out of 45 HCC biopsy specimens could establish PDX lines for an overall success rate of 44%. Expectantly, the majority came from male patients (n = 16; 80%). These were equally distributed among the four major HCC etiologies in the United States, with 8 (40%) from HCV, 6 (30%) from ALD, 1 (5%) from NAFLD, 2(10%) from HBV, 2 (10%) from alcohol with a secondary liver disease, and 1 (5%) with cryptogenic cirrhosis. These results on a small sample size suggest that HCC biopsy materials from a patient population in the United States can form PDX without a clear preference toward certain liver disease etiologies.

3.2 Human alpha1-antitrypsin is a serum marker of HCC PDX formation in mice

Most HCC tumor specimens have been implanted under the skin of mice, which allows for the detection of large PDX by palpation. When HCC are implanted into other anatomical sites palpation becomes insensitive for small tumors, imaging can be used but is operator-dependent, labor-intensive, and costly. We thus set out to test if human markers in mouse serum could identify animals that grew PDX. This would allow for easier PDX monitoring in non-subcutaneous implantation sites such as intrahepatic (IH) or subrenal capsule (SRC). Serum from mice that were implanted with HCC biopsies were screened for several human markers including albumin (hAlb), α₁-antitrypsin (hAAT), transferrin, and α-fetoprotein (hAFP). As illustrated by PDX1 (Figure 1A), hAAT became detectable in 3 out of 5 mice shortly after HCC implantation and rose over time along with hAlb, while hAFP (not shown) remained undetectable. PDX that released multiple human markers in mouse serum typically did so with similar ratios as illustrated by PDX3 (Figure 1B). Of the first 3 HCC biopsies that were transplanted into mice and that resulted in the rise of any human serum marker, all secreted hAAT. Other markers were inconsistently detectable. From then on serum hAAT was used as a marker for successful PDX formation. Indeed, all mice that contained macroscopic tumors showed rising hAAT levels. And conversely, of mice that were transplanted with HCC biopsy materials and that had unquantifiable (<50 ng/ml) hAAT levels for up to 6 months after surgery, none were found to have a macroscopic PDX. This suggests that serum hAAT, which historically has been investigated as a clinical HCC marker,³² is an effective tool to monitor PDX formation.

Next, sonographic detection of PDX was explored in mice with rising hAAT serum levels. In mice with high hAAT levels (e.g., >1 mg/ml for PDX1), PDX in the SRC could be visualized by ultrasound as a hypoechoic mass adjacent to the left kidney (Figure 1C). However, PDX in mice with low serum hAAT levels could not convincingly be visualized by ultrasound either for SRC or IH implanted biopsy materials.

Finally, serum hAAT levels were compared to tumor size. For passaged PDX1 there was a correlation (r = 0.8, p = 0.003 by Pearson correlation coefficient) between hAAT serum levels and tumor weight (Figure 1D). After accounting for the clustering serum hAAT and tumor weight data from different PDX lines did not show such correlation, which was in line with the highly heterogeneous nature of these tumors (beta = −434.72, p = 0.258) (Figure 1E).

These data show that hAAT quantification in mouse serum can serve as an effective tool to screen for PDX formation, and that for an individual PDX line hAAT serum levels correlate with tumor weight.

3.3 Only severely immunodeficient mice allow for PDX formation from HCC biopsy material

Innate, humoral and T cell-mediated rejection all form  major immune barriers to xenotransplantation,³³ which has led to the widespread use of T and B cell-deficient mice such as the severe combined immunodeficient (SCID) and recombination activating gene knockout (Rag^−/−) strains to establish PDX.³⁴ We here aimed to test to what extent the immunodeficient background of recipient mice influenced PDX formation. Three immunodeficient strains were used for PDX formation¹: T and B cell deficient Rag2^−/− animals²; Rag2^−/− mice additionally lacking the interleukin-2 receptor gamma chain (Rag2^−/− Il2rg^null), which results in the absence of natural killer and innate lymphoid cells and some impaired myeloid functions³; animals that in addition to Rag1^−/− Il2rg^null were on the non-obese diabetic background (NOD Rag1^−/− Il2rg^null or NRG), which impairs macrophage xenorejection.³⁵ Forty-five biopsy specimens were implanted in these three immunodeficient strains and rising serum hAAT levels were used as a marker for PDX formation. Not a single mouse generated rising hAAT levels with 20 biopsies implanted into Rag2^−/− animals. This contrasted with 9 out of 12 (75%) forming a PDX in mice on the Rag2^−/− Il2rg^null and 7 out of 12 (63%) in NRG mice (Figure 2A). The penetrance of PDX formation, defined as the number of mice that showed rising hAAT levels after HCC biopsy implantation, was higher in animals on the Rag2^−/− Il2rg^null (32%) than NRG (25%) immunodeficient background, which was not statistically different (p = 0.564) after accounting for the clustering from the different tumors (Figure 2B). Given the limited numbers of PDX there was no association between the immunodeficiency and HCC disease etiology. Mildly immunodeficient Rag2^−/− mice prevented PDX formation from all four major HCC etiologies in the U.S. (HCV, ALD, NAFLD, and HBV) whereas Rag2^−/− Il2rg^null and NRG mice allowed for PDX establishment from these four etiologies (Table 1). Of the 20 HCC specimens that could form PDX, five biopsy specimens were implanted in both Rag2^−/− Il2rg^null and NRG mice. Four grew PDX in both (HCC3 is an example of these four, Figure 2C) whereas one formed PDX only in the NRG immunodeficient background. These data show that the Rag2^−/− Il2rg^null immunodeficiency is minimally required to successfully establish PDX from HCC biopsy materials with no benefit of the NOD background.

3.4 The HCC biopsy implantation site affects PDX formation rate

Most PDX have been established by implanting HCC tissues subcutaneously^23-29 while only Zhu et al.³⁰ compared intrahepatic (IH) engraftment to subcutaneous implantation. Because of HCC’s arterial blood supply, we hypothesized that the subrenal capsule (SRC) that has historically been used for rapid revascularization of implanted tissue would improve PDX formation rates. To test this hypothesis HCC biopsy materials were implanted SRC and IH, and rising hAAT serum levels were used as a marker for successful PDX formation. Less immunodeficient Rag2^−/− mice were excluded from these analyses since this recipient strain precluded all PDX formation. Thirty HCC biopsy specimens were transplanted SRC and 22 biopsy specimens IH into mice on the Rag2^−/− Il2rg^null and NRG immunodeficient backgrounds. Overall PDX formation rates appeared higher with SRC (57%) than IH (30%) implantation, which did not reach statistical significance (Figure 3A). Penetrance, defined as how many mice formed PDX after HCC implantation, was indeed higher SRC (28%) than IH (9%) (Figure 3B). Twenty-two biopsy specimens were transplanted SRC in mice and side-by-side IH in other mice. Like the overall numbers, the SRC location trended to be superior for PDX formation (Figure 3C,D) while only four established PDX in both anatomical locations (Figure 3E). For the HCC biopsies that formed PDX in both locations no differences in hAAT kinetics were observed (not shown). Given the small number of PDX lines, there was no clear pattern between HCC disease etiology and location. These data show that HCC biopsy material forms PDX at a 2-fold higher rate under the renal capsule than in the liver, resulting in an overall 57% success rate in severely immunodeficient mice.

3.5 Few HCC require murine liver injury for PDX formation

Human hepatocytes can proliferate in response to signals provided by the damaged and/or failing mouse liver, which facilitates the creation of liver chimeric mice. We hypothesized that moderately and well-differentiated HCC, which are more associated with HCV and ALD than with HBV, would benefit from murine liver injury to form PDX. To test this hypothesis, we implanted HCC biopsy material into immunodeficient Fah^−/− mice that underwent liver injury by intermittent withdrawal of the protective drug nitisinone.³⁶ These liver injury mice were compared to immunodeficient Fah^−/− mice maintained on stable nitisinone or NRG mice without liver injury. We here analyzed the 18 HCC biopsies were implanted both in mice with and without liver injury, excluding Rag2^−/− recipients and combining SRC and IH implantation sites. PDX formed in 13/18 (72.2%) in mice with liver injury and 11/18 (61.1%) in animals without liver injury (p = 0.21) as defined by rising hAAT serum levels (Figure 4A). The penetrance was also numerically but not statistically significantly higher in mice with liver injury (38%) than mice without liver injury (25%) (Figure 4B). Two biopsies, one from a moderately differentiated HCC caused by HCV and another from a moderately differentiated ALD-associated HCC formed PDX in mice with liver injury but not in the same immunodeficient Fah^−/− mice kept on continuous nitisinone to prevent liver injury (Figure 4C). A third biopsy from a well-to-moderately differentiated HCC caused by ALD formed PDX in two out of four mice with liver injury and, only 4 months later, in a syngeneic mouse kept on continuous nitisinone without liver injury (Figure 4D).

These findings show that there is little overall benefit using Fah^−/− liver injury mice to establish PDX, yet some moderately and well-differentiated HCC may better form PDX using this approach.

3.6 Passaged PDX are less sensitive to anatomical and immunodeficiency restrictions

Once PDX have formed from HCC tissues they generally can readily be passaged into larger cohorts of animals,^{23, 28, 30} likely because subsets of tumor clones have been selected that are better adapted to the mouse environment.³⁷ We serially transplanted 12 PDX lines, 9 of which resulted in rising hAAT serum levels (75%). The penetrance, defined as the number of mice with rising hAAT levels after receiving a passaged PDX, was 85/146 (58%) and contrasted with the lower penetrance of HCC biopsy materials (Figure 2B). We mostly passaged SRC-derived PDX lines, that showed a modest preference for implantation site. Of the 9 PDX lines that could be passaged, SRC-derived tumors that were implanted SRC showed rising hAAT levels in 63/77 mice (82%) compared to 12/29 mice (41%) in which these were implanted IH (Figure 5A). This higher penetrance SRC was illustrated by PDX4, in which 4/4 SRC passaged PDX and only 1/3 IH passaged tumors resulted in rising hAAT levels (Figure 5B). Only 3 PDX that grew IH were passaged into new recipients. These PDX could be passaged in 2/6 mice (33%) SRC and in 4/18 mice (22%) IH. These data show that PDX established SRC were more efficiently passaged by SRC than IH implantation, while the reverse may not hold true.

For other cancers, serial PDX passage resulted in the selection of clones that increasingly lose the genetic complexity of the original tumor.³⁸ To create a repository of low-passage PDX that will be useful for future translational studies, non-passaged PDX would need to be cryopreserved in a way that allows for efficient engraftment after cryo-recovery. To test how cryopreservation affected engraftment efficiency and kinetics, we transplanted PDX1 that had been cryopreserved for 6 weeks in liquid nitrogen. Cryo-recovered PDX pieces were implanted in the SRC of 14 NRG mice and compared to 20 NRG mice that had received the same PDX in the SRC without cryopreservation. Thirty-six days after the respective surgeries hAAT levels in freshly transplanted PDX were 19-fold higher than in mice that received cryopreserved tissues of comparable weight (Figure 5/span > C). Nevertheless, all cryopreserved tissues showed rising hAAT levels with minimal variation, illustrating that despite cryopreservation this PDX was able to survive the passaging process.

Finally, we tested whether PDX could be passaged into less immunodeficient recipients. Having failed to establish any PDX from HCC biopsy in T and B cell-deficient Rag2^−/− mice, we passaged one PDX into Rag2^−/− and four PDX lines into Foxn1^nu (nude) mice that lack only T cells. Interestingly, one out of four Rag2^−/− mice showed rising hAAT levels. Two of four PDX lines could be successfully passaged in nude mice. PDX3, which had been passaged through the liver of an NRG mouse, was subsequently able to grow in livers of nude mice with similar hAAT kinetics as in NRG animals (Figure 5D). Upon harvest, solid tumors were observed in the livers of these nude animals (Figure 5E). When these PDX3 from nude livers were further passaged into wild-type Balb/c mice no rising hAAT levels were observed in any recipients, suggesting that murine T cells retained the ability to reject this tumor.

These combined data show that a majority of PDX can be passaged with a persistent preference for the SRC space. Furthermore, some passaged PDX lines can grow in less immunodeficient mouse strains.

3.7 PDX morphologically resemble the HCC biopsy

We finally set out to compare PDX histology to clinical histology. Eight PDX lines were recovered and processed for H&E staining. We found that 10 out of 10 (100%) PDX tumors retrieved from 8 HCC primary tumors resembled histopathological characteristics of original human tumor. All tumors were conventional HCC, belonging to the following WHO histologic types: trabecular (2/8 cases), macro-trabecular (3/8), and solid or compact (3/8). Comparative evaluation of the paired biopsy and PDX samples revealed that the key histological characteristics seen in the biopsies for each individual tumor were recapitulated by the PDX (Figure 6). Specific histological traits seen in paired biopsy and PDX samples are outlined in Table 2. These data show that PDX faithfully recapitulate histological features of the original HCC.