The Use of the Protease Inhibitor, Saquinavir, to Treat Anal Cancer Spheroids Derived from HPV Transgenic Mice

Abstract

BACKGROUND:

Anal cancer is associated with high-risk human papillomavirus infection and oncoprotein expression. We have identified several protease inhibitors, used to treat HIV, that decrease oncogene expression.

OBJECTIVE:

The aim of this project is to determine whether the protease inhibitor, Saquinavir, results in a treatment response in anal cancer spheroids.

DESIGN:

K14E6/E7 transgenic mice (n=5), which express HPV16 oncoproteins E6 and E7 in their epithelium, were treated topically at the anus with carcinogen, 7,12 dimethylbenz[a]anthracene, to promote anal tumor growth. Tumors were excised and digested, and cells were plated. The tumor cells form 3D multicellular aggregates, known as spheroids.

SETTINGS:

This study was performed in an American Association for Accreditation of Laboratory Animal Care approved facility.

INTERVENTIONS:

Spheroids were placed in treatment groups: no treatment, vehicle (dimethyl sulfoxide), and 15 μM Saquinavir. Spheroids were imaged immediately prior to treatment and 24-hours post-treatment.

MAIN OUTCOME MEASURES:

Spheroid diameters were measured using ImageJ and mean percent reduction was calculated for each spheroid to determine treatment effect on spheroid growth. Analysis of variance using pairwise comparisons were performed with Fisher’s protected least significant difference tests.

RESULTS:

No treatment (n=119 spheroids) and vehicle (n=126 spheroids) groups demonstrated an increase in spheroid diameter over the treatment period. In contrast, spheroids treated with Saquinavir (n=151 spheroids), demonstrated a statistically significant percent reduction compared to no treatment (p value < 0.0001) and vehicle (p value = 0.002) groups.

LIMITATIONS:

A limitation of this data is that some human error is likely present given that images were analyzed by three different scientists.

CONCLUSIONS:

Saquinavir leads to a statistically significant percent reduction in mice anal tumor spheroid growth ex-vivo when compared to control groups. Protease inhibitor therapy may be an effective treatment or adjuvant therapy to the Nigro protocol to promote anal cancer tumor regression. See Video Abstract at http://links.lww.com/DCR/Bxxx.

INTRODUCTION

Anal cancer is associated with high-risk human papillomavirus (HPV) infection (HPV 16 and 18).¹ Approximately 9,000 patients are diagnosed with anal cancer annually in the United States.² The incidence of anal cancer continues to grow despite HPV vaccination and screening for precancerous lesions.³ Persistent infection with high-risk HPV strains, results in constitutive activation of HPV oncoproteins E6 and E7, which can lead to precancerous lesions and ultimately progress to anal cancer. Anal carcinogenesis is associated with the inhibitory effects of HPV oncoproteins on well-known tumor suppressor genes, p53 and pRb.⁴

Our study looks at a protease inhibitor, Saquinavir, an FDA-approved drug used to treat human immunodeficiency virus (HIV), as a possible novel treatment for anal cancer. It has been demonstrated that protease inhibitors are generally well-tolerated by patients and have been successful in the treatment of HPV-associated disease. The efficacy of these drugs is attributed to the suppression of the E6 oncoprotein.^5,6

In general, current treatments for anal cancer are poorly tolerated by patients due to a significant side effect profile. There have not been significant improvements in anal cancer treatment in almost 50 years: Dr. Norman Nigro developed the Nigro protocol in 1974. This protocol involves the use of 5-fluorouracil or capecitabine, mitomycin-C, and radiation therapy. Side effects related to treatment of anal cancer are debilitating in nature including acute complications such as diarrhea, skin irritation and breakdown, and long-term issues with bowel, bladder, and sexual dysfunction.⁷ Therefore, there is a need for an effective and well-tolerated treatment for anal cancer.

Because of poor patient tolerance of currently available treatments and the difficulty performing clinical trials in this population due to limited patient numbers, an ex-vivo spheroid culture model provides unique advantages for studying anal cancer treatment. Spheroids are three- dimensional cellular aggregates that represent tumor behavior.^8,9 For our purposes, spheroids are obtained directly from mice anal tumors and are used to study tumor growth and drug therapy responses. This is a reproducible model that can predict in-vivo response to treatment in an ex-vivo manner. We hypothesized that Saquinavir, a protease inhibitor, would reduce growth of mice anal tumor cells in spheroid culture and therefore may be an effective therapy for anal cancer.

MATERIALS AND METHODS

Mice

Male and female K14E6/E7 transgenic mice were generated as previously described by Stelzer et al., 2010. These mice express high-risk HPV strain (HPV16) oncoproteins, E6 and E7, in their epithelium. K14E6/E7 mice reliably develop spontaneous anal dysplasia that can progress to anal cancer, in a manner that is comparable to anal carcinogenesis in humans.¹⁰

All mice were housed in an American Association for Accreditation of Laboratory Animal Care approved facility, the Wisconsin Institute for Medical Research (WIMR) Animal Care Facility. This study was performed in accordance with approved Institutional Animal Care under approved animal protocol M006333.

7,12 Dimethylbenz[a]anthracene (DMBA) Treatment

Mice were treated with the carcinogen, 7,12 dimethylbenz[a]anthracene ((DMBA), Sigma Aldrich, Saint Louis, MO, USA) to ensure anal tumor development. A 0.12 μmol DMBA solution in 60% acetone (Avanton, Radnor, PA, USA) and 40% dimethyl sulfoxide ((DMSO), Thermo Fisher Scientific, Waltham, MA, USA) was applied to mice topically at the anus, once weekly until anal tumors developed.¹¹ Animals were monitored weekly for overt anal tumor growth and local side effects. When mouse tumors reached approximately, 5 mm by 5 mm or a tumor volume of 65.4 mm³, mice were euthanized in a carbon dioxide tank followed by cervical dislocation. Following euthanasia, mice anal tumors were sharply excised.

Spheroid Creation/Treatments

Anal tumors were individually rinsed in a 1x phosphate buffered saline ([PBS], Corning, Tewksbury, MA, USA) solution in a sterile 5 mL tube and placed on ice. Anal tumor digestion, cell isolation, plating, and spheroid maintenance were performed as described in Pasch et al., 2019 with minimal modifications.⁹ Figure 1 provides a flow diagram of our methodology. Please see Supplementary Materials for more information.

Figure 1.

Flow diagram of our methodology. Transgenic K14E6/E7 mice began weekly topical application of carcinogen until anal tumors reached size threshold (5x5 mm). Mice were euthanized and anal tumors were harvested and digested. Cells were prepared, plated, and fed. Anal tumor spheroids underwent initial imaging, were randomized into treatment groups, and were treated with drug. Spheroids were re-imaged 24 hours after treatment. Please see Supplementary Materials at https://links.lww.com/DCR/CXXfor more information. Permission to publish granted by Bio Render.

In order to establish a final Saquinavir treatment concentration, a preliminary plate of spheroids was treated with Saquinavir (Sigma Aldrich, Saint Louis, MO, USA) dissolved in DMSO to obtain a final concentration per well: 5 μM, 10 μM or 15 μM Saquinavir. Spheroids were treated by pipetting varying volumes of a 5 mM Saquinavir DMSO solution into the respective well’s media to achieve the final desired concentration. The final concentration of 15 μM Saquinavir was determined based on the lowest concentration that resulted in the greatest treatment effect (Fig. 2). Culture plate wells that contained viable spheroids were randomized into treatment groups to achieve an approximately equal number of spheroids per treatment group. Final treatment groups included: control (no treatment), vehicle (DMSO), and 15 μM Saquinavir dissolved in DMSO. DMSO was the diluent for Saquinavir and therefore used as a control for this experiment. An equal volume of DMSO to match the volume of Saquinavir dissolved in DMSO in the Saquinavir group was used for the vehicle group.

Figure 2.

Dose response curve to varying concentrations of Saquinavir used to determine concentration utilized for anal spheroid treatment (N = 2 mice per treatment group). Based on this treatment, the dose of 15 μM of Saquinavir was selected for treatments of spheroids as it was the dose with the largest percent change in spheroid diameter.

Spheroid Imaging

Imaging was performed immediately prior to treatment and again 24 hours post-treatment. A 24 hour timeframe was chosen based on the fact that this was the time of greatest response noted in our dose response curve. Additionally, higher rates of well contamination are noted with longer timepoints. All images were taken using a Nikon Eclipse Ti-S inverted microscope with the NIS elements AR 5.20.00, 64-bit software, and a 4x objective lens. Each well containing spheroids was imaged, ensuring that individual spheroids were in focus for their respective photographs. In order to ensure accuracy in imaging over the 24 hour treatment period, well mapping was utilized allowing us to save position of the microscope for each well and for each image of spheroids. This allows for images of spheroid to be captured at the same location in the well throughout the experiment.

Image Analysis

Images were analyzed using ImageJ Version 2.10/1.53c. Each spheroid diameter measurement was taken at three distinct points of the spheroid to account for variability in spheroid shape. The three diameter measurements per spheroid were averaged and percent reduction was calculated. Percent reduction was calculated using the formula = [(Average Pre-treatment Diameter - Average Post-treatment Diameter) / Average Pre-treatment Diameter] × 100].

Statistical Analysis

For the purpose of this project, unique spheroids were treated as distinct cell lines. Data underwent analysis of variance testing with pairwise comparisons performed with Fisher’s protected least significant difference tests using SAS Version 9.4 (SAS Institute, Inc., Cary, NC, USA).

RESULTS

Dose Response Curve for SQV

K14E6/E7 anal cancer spheroids were treated with various concentrations of Saquinavir as seen in Figure 2 (two spheroids per treatment concentration). Based on this treatment, the dose of 15 μM of Saquinavir was selected for treatments of spheroids as it was the dose with the largest percent change in spheroid diameter.

Tumor Development

A total of five K14E6/E7 mice (two male and three females) reached tumor threshold (approximately 5 mm by 5 mm or a tumor volume of 65.4 mm²) within our study period. Mouse tumors were harvested and digested for cell culture. The cells from each mouse were split into each of the experimental conditions in replicates and the outputs in terms of spheroids from each mouse ranged from 24-209 spheroids. The number (n) of spheroids depends on the size of the tumor harvested and the number of viable spheroids.

Spheroid treatment

Upon analysis, there was a statistically significant association between treatment group and average percent reduction (p < 0.0001). Figure 3 demonstrates representative spheroids from each treatment group pre-treatment and 24 hours following treatment. As demonstrated in Figure 4, the no treatment group (n = 119 total spheroids) demonstrated an increase in spheroid size over the 24-hour treatment period with a mean percent increase of 7.55%. The vehicle (n = 126 total spheroids) group also demonstrated a 4.68% mean increase in spheroid size over the 24-hour treatment period. In contrast, spheroids treated with Saquinavir (n = 151 total spheroids), demonstrated a statistically significant percent reduction or decrease in size at 0.24% when compared to no treatment (p < 0.0001) and vehicle (p = 0.002) groups. There was no statistically significant difference between no treatment and vehicle groups (p = 0.083).

Figure 3.

Representative spheroids from each treatment group pre-treatment and 24 hours following treatment. A & B represent the no treatment group. C represents vehicle group pre-treatment. D represents vehicle group 24 hours post-treatment. E represents Saquinavir group pre-treatment and F represents Saquinavir group 24 hours post-treatment. These images were taken with a 4x objective.

Figure 4.

Mean percent reduction of all spheroid average diameters in each treatment group ± standard error of the mean (SEM). Percent reduction was calculated using formula = [(Average Pre-treatment Diameters - Average Post-treatment Diameters) / Average Pre-treatment Diameters] × 100]. The no treatment group has an n of 119 total spheroid diameters (each diameter measurement is the average of three diameter measurements per spheroid to account for variability in shape) vehicle with an n of 126 total spheroids diameters, and Saquinavir with an n of 151 total spheroids diameters. Figure 4 demonstrates that Saquinavir led to a positive percent reduction or decrease in spheroid size whereas the no treatment and vehicle groups led to a negative percent reduction or increase in spheroid size. Significance was assigned as *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001.

DISCUSSION

Saquinavir leads to statistically significant reduction in mice anal tumor spheroid growth when compared to control groups (no treatment and vehicle). Therefore, Saquinavir may be an effective treatment or adjuvant therapy to promote anal cancer tumor regression. As expected, there was not a significant difference between no treatment and vehicle (DMSO) groups, though DMSO showed only a slightly lower percent increase in comparison to the no treatment group. These results support a growing body of literature supporting protease inhibitors as a potential therapy for HPV-induced cancers. Though there is not yet published literature to support protease inhibitor treatment for anal cancer, protease inhibitors were shown to be effective in the treatment of HPV-induced head, neck, and cervical cancers through decreasing carcinogenesis and as a radiosensitizer.^12-14 Research supports that Saquinavir functions by inhibiting HPV-associated oncoprotein E6 thus preventing E6-mediated inhibition of tumor suppressor gene p53.^5,6

Strengths of this study include the use of an already FDA-approved drug, Saquinavir, in our research. Though protease inhibitors are currently approved for treatment of HIV, the use of market-available drugs is ideal for both clinicians and patients. An additional strength is that spheroids are a reproducible and proven culture model for replicating in-vivo tumor response.⁹

Limitations of this data include a small sample size of only five mice tumors. However, we prefer to treat distinct spheroids as others treat cell lines, since each tumor can produce hundreds of unique spheroids (n = 396 spheroids). Another limitation of this study is that we did not collect cell lysates or RNA to study targets of protease inhibitors. Due to supply chain issues during our study period, we had to change from Matrigel to Extracellular Matrix (ECM) gel and found equivalent results. Additionally, some human error is likely present, given that images were analyzed and measured by three different scientists and there is significant variability in spheroid dimensions.

Our research has important implications for those suffering from anal cancer. An effective and better tolerated treatment for anal cancer would significantly improve the quality of life of patients suffering from adverse drug effects and possibly prevent the need for major abdominal and pelvic surgery.

Future directions include the use of optical metabolic imaging (OMI) to provide more accurate measurement of spheroid growth and/or reduction following treatments. OMI utilizes spheroid metabolism in order to more accurately track changes in spheroid size. Additional work conducted by our group includes studying the ability of topical protease inhibitors to prevent anal carcinogenesis in a transgenic mouse model and in an immunodeficient NOD scid gamma (NSG) mouse model infected with Mus musculus papillomavirus (MmuPV1), to determine if protease inhibitors are effective in preventing the progression of anal dysplasia to anal cancer. Through these mouse models, we are able to study targets of protease inhibitor inhibition with immunohistochemistry and immunofluorescence to better understand Saquinavir’s drug effects. Future work includes a Phase I clinical trial for topical protease inhibitors, for which our lab has secured funding to determine safe and tolerable dosing for topical protease inhibitors in humans.