Development of Human-Derived Cell Culture Lines for Recurrent Respiratory Papillomatosis

Abstract

Recurrent respiratory papillomatosis (RRP) is mainly caused by Human Papillomavirus (HPV) 6 and 11. While various adjuvant therapies have been reported, no effective therapy has been documented to universally “cure” this disease. In the era of precision medicine, it would be valuable to identify effective intervention based on drug sensitivity testing and/or molecular analysis. It is essential to be able to successfully carry out in vitro culture and expand tumor cells directly from patients to accomplish this goal. Here we report the result of successful culture of HPV-infected cell lines (successful rate 70%, 9/13) that express the E6/E7 RNA transcript, using pathologic tissue biopsies from patients treated at our institution. The availability of such a system would enable ex vivo therapeutic testing and disease modeling.

Introduction

Recurrent respiratory papillomatosis (RRP) is a rare disease with an estimated incidence of 4.3 and 1.8 cases per 100,000 in those ≤ 14 years and ≥15, respectively. Approximately, 1500–2500 new pediatric cases occur in the U.S. each year. RRP is characterized by multiple exophytic wart-like lesions within the respiratory tract, most commonly involving the larynx, especially the vocal cords ⁽¹⁾. Low-risk human papillomavirus (HPV) types, 6 and 11 account for more than 90% of all RRP cases ⁽¹⁾. This disease poses a challenge to the patient, family and treating physicians ^{(2, 3)}. According to the National Registry of Children with RRP, patients undergo an average of 4.4 (0.2–19.3) procedures per year ⁽⁴⁾. Many patients require operative intervention for years and may undergo 80–100 procedures. No universally effective drug has been documented ⁽⁵⁾. It would be valuable to identify individualized therapy based on drug sensitivity test and/or molecular analysis, especially for refractory RRP. It is essential to have an in vitro model for culturing tumor cells directly from patients to accomplish this goal.

The generation of human-derived cell culture lines for RRP is a nuanced laboratory technique and has been previously described in clinicopathologic and genetic profiling of recalcitrant and aggressive disease in a single patient ⁽⁶⁾. However, it is not clear whether similar approach could work for more typical and less aggressive tumors, and whether the HPV genomes would be lost if cell lines were established. Here we report successful development of a disease model with 9 robust HPV-infected cell lines using pathologic tissue biopsies from patients treated at our institution.

Methods

Tissues and cell culture

Informed consent was obtained per institutional human subject protections requirements (UCSD IRB#151676). Fresh papilloma specimens were collected from patients with active papilloma (Table 1) undergoing surgical debridement and control specimens were obtained from children undergoing routine tonsillectomy.

Table 1.

Specimen details

Case Number	HPV Type	In vitro Cell growth	Number of surgeries	Sex	Ages	Diagnosis
1	6	Y	70	F	15.3	Papillomatosis of larynx
2	6	Y	50	M	16.5	Papillomatosis of larynx
3	11	N	68	M	12.5	Papillomatosis of trachea
4	6	N	29	F	25.5	Papillomatosis of larynx
5	11	Y	92	F	23.5	Papilloma of larynx
6	6	Y	21	F	4.7	Recurrent glottic respiratory papillomatosis
7	6	Y	7	F	18.6	Laryngeal papillomatosis
8	11	Y	6	M	2.3	Recurrent respiratory papillomatosis
9	11	Y	3	M	16.4	Papilloma of nasopharynx
10	6	N	21	F	3.5	Recurrent respiratory papillomatosis
11	11	N	21	F	11.0	Recurrent glottic respiratory papillomatosis
12	11	Y	29	F	18.8	Papilloma of nose
13	6	Y	47	M	22.2	Papillomatosis of larynx

Tissue samples were minced and digested with Dispase II (5mg/ml; Roche Life Science) and then Trypsin. Cells were cultured in EpiLife® Medium (Gibco, ThermoFisher, MA, USA) supplemented with Human Keratinocyte Growth Supplement (Gibco); Bovine Pituitary Extract (Gibco); 10–40 ng/ml Recombinant Human EGF Protein (R&D systems, Minneapolis, MN); 10–30 ng/ml Recombinant Human FGF Basic Protein (R&D systems) and addition of 5–10 umol/L Y-27632 (Enzo Life Sciences, Farmingdale, NY, USA) without feeder cells to facilitate downstream analyses.

Immunofluorescence

Cells grown on coverslips were fixed with 4% formaldehyde, blocked with 3% BSA, incubated in mouse anti-cytokeratin 5 (ab17130; Abcam, Cambridge, MA, USA), rabbit anti-cytokeratin 8+18 (ab53280; Abcam), mouse immunoglobulin (SC-2025; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or rabbit immunoglobulin (#2729; Cell signaling Technology, Beverly, MA, USA), detected with Alexa Fluor 488 anti-mouse IgG ( Life Technologies, Carlsbad, CA, USA) or Alexa Fluor 568 anti-rabbit antibody ( Life Technologies) and mounted with ProLong Gold Antifade Mountant with DAPI (Life Technologies). To demonstrate the specificity of the immunofluorescent staining, cells were also stained with secondary antibodies without primary antibodies⁽⁷⁾. Images were captured on a Nikon A1R confocal microscope with lasers at 405, 488, and 561 nm excitation wavelengths using a 60X 1.4 NA objective at a resolution of 0.2 microns per pixel (Nikon, Melville, New York).

Nucleic acid Analysis

All of the clinical samples were subject to HPV typing (13 high-risk and 24 low/intermediate genotypes) by PCR L1 gene for reverse blot hybridization with a linear array (Quest Diagnostics, San Juan Capistrano, CA, USA).

For in-house E6/E7 based typing, total RNA and DNA were isolated from the samples using RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and Quick-DNA Universal Kit (Zymo research, Irvine, CA, USA), respectively. Purified RNA was reverse-transcribed into cDNA using SuperScript VILO cDNA Synthesis System (Invitrogen, Carlsbad, CA) with random primer. The PCR reactions for the detection of the genomic DNA and E6 mRNA of HPV contained 2 μl of DNA/cDNA, 200 μM of dNTP, 1.5 mM MgCl₂, I U Taq DNA polymerase, and 200 nM of each primers ⁽⁸⁾. For the multiplex PCR to genotype HPV 6 and 11, one HPV6/11 universal forward primer⁽⁸⁾ and two genotype-specific reverse primers (HPV6: 5’-TTA TGA ACC GTG CCT TGG TTA G-3’; HPV11: 5’-CAA CGA CCC TTC CAC TGG TTA-3’) were used. Two sets of primers were used to genotype HPV16⁽⁸⁾ and HPV18⁽⁹⁾. The PCR products were resolved in 2% agarose gels and stained with ethidium bromide.

Results

All of the clinical diagnosis of RRP was validated by pathological examination of biopsied tumor samples (Figure 1A). Thirteen patients (Table 1) were recruited and 9 of samples were successfully cultured and expanded. Three lines of evidence suggest that these cell lines were derived from RRP tumor cells. First, the cell morphology shows typical cobblestone-like epithelial cells (Figure 1B). Second, these cells were positive for epithelial cell markers, cytokeratins (Figure 1B). Third, these cell lines were positive for either HPV 6 or HPV 11 (see below).

Figure 1: Recurrent respiratory papillomatosis (RRP) and Tonsil cells in culture.

(A) Pathological diagnosis. (B) Cultured cell morphology (phase contrast) (C) Cytokeratin staining of cultured RRP cells. (D) Cytokeratin staining of tonsil cells. DIC, Differential Interference Contrast; IF, immunofluorescence.

In addition, 19 of 23 tonsil cell lines were established from patients with benign diseases.

While all of the clinical specimens from RRP debulking were positive for either HPV6 or 11 (Table 1) using a genotyping array for 37 HPV types, it was not clear whether cultured cell lines would lose HPV episomes. E6 specific primers were used for HPV 6/11 typing and for detecting viral RNA expression in cells. Representative results of 2 tonsil (negative control) and 3 RRP cell lines are presented in Figure 2A. Our analyses reveal that HPV DNA (lane 3, 6, 7, 10) was present and the E6/E7 mRNA transcript (lane 5, 8, 12) was expressed in cultured cells. No HPV DNA was detectable in the cultured medium (lane 5, 9, 13), suggesting no viral particle was produced from cell culture. No HPV 16 and 18 were detected in all of the cell lines (Figure 2B and 2C), which is consistent with clinical genotyping result.

Figure 2: Human papillomavirus (HPV) analysis.

(A) The Presence of HPV DNA (Cell, DNA) and culture medium (Sup, DNA), and the expression of E6/E7 RNA (Cell, RNA) in cells are indicated for two tonsil (Lane 1–2) and three RRP (Lane 3–13). Clinically typed HPV6 (Lane 14) and HPV11 (Lane 15) positive RRP tissues were used as positive controls. Water (Lane 16) was used as a negative control for PCR amplification. (B) HPV 16 PCR. (C) HPV 18 PCR.

Discussion

In this short communication, we report a high success rate (69.2%, 9/13) of primary tumor cell culture directly from patient biopsy specimens by applying a modified protocol⁽⁶⁾. The same protocol also works efficiently for culturing tonsil epithelial cells, which are used for control experiments. Characterizing some of these patient-derived cells revealed persistence of E6/E7 expression. However, no viral particle was detectable in the culture medium. This is consistent with the fact that HPV viral cycle is tightly regulated by host cell differentiation ⁽¹⁰⁾ and the Rho kinase inhibitor treated cells were conditionally reprogrammed to a less differentiated state ⁽¹¹⁾. Nevertheless, cell differentiation can be restored when a proper medium was used without this inhibitor ⁽¹¹⁾. Therefore, specialized protocols can be developed to accommodate individual needs. Further development is ongoing; having been successful at growing these cell cultures we have begun to test them with various chemotherapeutic/antimicrobial compounds beginning with the antiviral agent Cidofovir. It would also enable the development of potentially curative adoptive cell immunotherapy for RRP, given the promising report of applying HPV targeted tumor infiltrating lymphocyte to treat patients with refractory cervical cancers ⁽¹²⁾.