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Abstract

Background

Sarcomatoid (and rhabdoid) dedifferentiation can occur in ∼5% of renal cell carcinomas (sRCC), a finding with significant therapeutic implications. sRCC is associated with increased aggressiveness, resistance to conventional targeted therapies, and increased sensitivity to immune checkpoint inhibitors.

Objective

There are no preclinical models for sRCC that can be used in research to better understand this disease. The pig has similar size, anatomy, immune system, and genetics, that can be employed to evaluate procedures or tumor specific drugs. We report on the creation of a large animal porcine model for sRCC.

Materials

In eight Oncopigs, a Cre-recombinase gene adenoviral vector (AdCre) was incubated with renal tissue obtained from an ultrasound (US) percutaneous biopsy and reinjected into the renal cortex. US was performed to assess growth and to obtain tumor tissue for pathologic and immunohistochemistry evaluation.

Results

Three weeks post inoculation renal tumors were successfully formed in 28 out of 32 sites (88%). Mean tumor size by imaging was 1.6 × 1.1 cm (range: 0.4–3.9 cm longest axis). Pigs remained clinically healthy up to 25 days after inoculation. On histology, tumors consisted of foci of infiltrating sarcomatoid and rhabdoid cells in a background of marked acute and chronic inflammation. The neoplastic cells showed positive immunoreactivity for PAX8, cytokeratin AE1/AE3 supporting a renal tubular origin. These cells were diffusely positive for p53 and showed high ki-67 (20–30%), and cleaved caspase 3 (20–30%) expression.

Conclusion

Rapid growing poorly differentiated neoplasms associated with a marked inflammatory reaction that have phenotypic and immunohistochemical features of sRCC were successfully developed. These may be suitable to study the response to local and systemic therapies.

Keywords

renal cell carcinoma sarcomatoid renal cell carcinoma swine transgenic animals large animal model

Introduction

Renal cell carcinoma (RCC) accounts for 90% of kidney cancers.^1,2 It is estimated that over 431,000 new cases of RCC are diagnosed each year worldwide and 200,000 deaths.³ Dedifferentiation in RCC, an infrequent event, is observed as two phenotypes, sarcomatoid (sRCC) or rhabdoid (rRCC) change and may occur in the setting of any RCC histology.^4,5 Rhabdoid change, though less known, has many parallels to sarcomatoid change. Sarcomatoid and rhabdoid dedifferentiation (s/rRCC) both presage inferior survival, are considered grade 4 at diagnosis, and have therapeutic implications.⁶

The use of conventional chemotherapies to treat s/rRCC were particularly underwhelming and ineffective.^7–9 Research in the field of oncology has enhanced our knowledge concerning the genetic, molecular, and immunogenic changes which occur within tumors resulting in the development of new targeted drugs for precision treatments. Presence of s/rRCC confers inferior response to first line angiogenesis targeted therapies, with a median survival after diagnosis of only around one year.¹⁰ Immune checkpoint therapy with the goal of inhibiting the interaction between immune cells and tumor to impede tumor tolerance to the immune system by targeting programmed death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte associated protein 4 (CTLA-4) with nivolumab plus ipilimumab has significantly improved outcomes for patients with s/rRCC, particularly in patients with increased expression of the target receptor and an inflamed tumor microenvironment.^11–13 Despite the therapeutic implications, the biologic process underpinning s/rRCC are still not well understood and no exact immunocompetent animal models to investigate this disease exist to date. There is an urgent clinical need to develop and utilize an animal model of s/rRCC that can replicate human cancer to evaluate new and combined treatments to improve outcomes.

Historically, murine models have played a major role in our understanding of cancer and the role specific genes and gene mutations have in the development and progression of cancer. However, due to vast differences between humans and rodents, the ability to model a complex disease such as cancer and quickly translate results to clinical practice is quite limited.¹⁴ New cancer therapies developed in rodents have a high failure rate when translated to humans.¹⁵ When compared to humans, pigs are an archetype large animal models to mimic diseases such as cancer due to the similarities in size, anatomy, metabolism, genetics and immune system compared to humans.¹⁶ Research in pigs has already been shown to be more predictive of therapeutic treatments in humans than rodents.¹⁷ Compared to cellular or small animal models of RCC, a porcine model has the potential to shorten the timeline for preclinical to clinical translation and substantially reduce the total cost of research. Additionally, the small size of rodents prohibits the testing of device-based tools and procedural techniques widely employed in clinical practice. Novel minimally invasive therapies, especially in the fields of interventional radiology, urology, and radiation oncology can be developed and improved by testing in a pig RCC model.

A transgenic oncopig model has been established and already used in the production of site-specific orthotopic tumors in the liver, lung, and pancreas.^18–20 Here we present the feasibility, pathologic, and immunohistochemical description of the development of a site specific porcine renal tumor in an oncopig.

Methods and materials

The Institutional Animal Care and Use Committee approved all research procedures. The animals’ housing facility was accredited by the AAALAC International and was in compliance with the United States Department of Agriculture Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals. Procedures were performed by a board-certified interventional radiologist.

Eight, nine-week-old Oncopigs (transgenic pigs with Cre-inducible TP53R167H and KRASG12D mutations) with a mean body weight of 25 kg were obtained from Sus clinicals Inc (Chicago, IL). The animals were allowed to acclimate to our animal facility for 5 days. Prior to any procedures the animals were fasted for 12 h. Each pig was sedated with an intramuscular injection of solution containing ketamine hydrochloride, acepromazine, and atropine sulfate. Anaesthesia was induced with isoflurane administered via mask. Once the pig was anaesthetised, an endotracheal tube was inserted, and anesthesia was maintained with isoflurane, nitrous oxide, and oxygen.

Tumor induction (in situ method)

Direct imaging of the kidney for accurate needle guidance and placement is required. For this experiment real-time ultrasound (US) guidance was used. An 18-gauge coaxial core biopsy needle set (Bard Mission, BD, Franklin Lakes, NJ) was placed into the renal cortex of the upper and lower poles of the right and left kidneys, a single, 2 cm long tissue sample was obtained through the coaxial needle using the biopsy device. The tissue sample was incubated at room temperature for 20 mins with an adenoviral vector (10^9 pfu Ad5CMVCre, University of Iowa Viral Vector Core) in phosphate-buffered saline that contained 15 mM calcium chloride. Calcium chloride added to adenovirus in phosphate-buffered saline results in co-precipitation of adenovirus and calcium phosphate, which improves viral transduction. The virus carries the Cre recombinase gene and activates TP53R167H and KRASG12D expression. Ex vivo incubation of the core biopsy with AdCre ensures that the renal epithelial cells are exposed to the vector. At this point, a slurry was fashioned from the virus and tissue mixed with Gelatin sponge (Gelfoam, Pfizer, Kalamazoo, MI) using a 3-way stopcock. Again, using image guidance, the initial coaxial needle that remained in place was advanced from the cortex to the medullary cortical junction of the kidney. The previously incubated slurry mixture was then percutaneously injected back into the kidney. Site selection was deep enough into the renal parenchyma to avoid leakage of injected material into the peritoneum and as far apart as possible (> 6 cm) so each inoculation site would form a distinct tumor mass. Additionally, the location was chosen to ensure safe and easy percutaneous access for subsequent tissue biopsy of the tumors after they formed.

Animals were clinically monitored every day though the entire follow-up period, particularly for any signs of pain, changes in fluid status, urine production, and blood in the urine. Blood samples were obtained at 3 weeks.

Imaging

Animals were placed under monitored anesthesia care for US imaging, which was performed on day 14 to monitor tumor growth. Pigs received general anesthesia with endotracheal intubation on day 21 with repeat US to assess the size, tumor imaging characteristics of the renal masses, and obtain tissue.

Pathology

Three weeks after inoculation, animals underwent image guided biopsy with real time US guidance, tumor samples were again obtained via coaxial technique. Samples were macroscopically examined, harvested, and fixed in 10% neutral buffered formalin. Following formalin fixation, sections of tumor were processed into paraffin blocks, and sectioned at 5-micron thickness. Haematoxylin and eosin (H&E)-stained sections were obtained and reviewed by genitourinary trained pathologists. Representative formalin-fixed paraffin-embedded tissue sections were immunolabeled with antibodies against 18 different markers used to diagnose RCC, for example CD10, 31, 68, 117, Carbonic anhydrase IX, PAX8, cytokeratin AE1/AE3, PDL-1, p53, MIB-1, Cathepsin K, Vimentin, and Caspase 3.

Results

Inoculation success

Investigating the previously described percutaneous in-situ approaches of tumor inoculation, our success rate for renal tumors was 28 out of 32 sites (87.5%). Tumors were imaged with US on days 14, 15, 21, 22, and 23 post inoculation. There were no complications from the tumor inoculation procedure, based on daily clinical evaluation and weekly US imaging. 71% of tumors were visualised at the earlier time point (14–15 days post injection) and 88% were seen at the later time point. All renal tumors appeared to be distinct solid masses with heterogeneous echogenicity on US, furthermore vascular flow was visualized on doppler evaluation. In 7% of animlas, tumors appeared to extend beyond the renal capsule and were partially exophytic. Average tumor size at 3 weeks post inoculation was 1.6 × 1.1 cm, in the long and short axis (range: 0.4–3.9 cm longest axis). All pigs remained clinically healthy during the duration of the follow-up period. There were no changes to the urine output, no blood was visually identified in the urine, and no changes were identified on creatinine from blood chemistry.

Renal tumor pathology

US-guided coaxial biopsy (18-gauge) samples were obtained from 6 animals (20 tumors). On histological examination of H&E-stained sections from the neoplastic renal and perirenal mass revealed well demarcated by infiltrating sheets of markedly atypical epithelioid cells. These cells were characterised by large nuclei with prominent nucleoli that varied from spindled to epithelioid shape and moderate to abundant eosinophilic cytoplasm, that at places pushed the nuclei to the periphery giving it a rhabdoid appearance. Tumor necrosis, mitosis, including atypical mitosis was frequently observed. No differentiated epithelial component was identified. The background stroma was associated with abundant mixed inflammatory infiltration consisting of lymphocytes, plasma cells, histiocytes, multinucleated giant cells and neutrophils. At places the inflammatory infiltrate was the dominant component present (Figures 1 and 2).

Figure 1.

Histology of neoplastic renal nodules after renal tumor inoculation in an oncopig. A and B) Representative low (40x) magnification H&E staining images demonstrating ill-defined tumor comprising of sheets of infiltrating markedly atypical epithelioid cells. C and D) Higher magnification (200x) reveals that the tumor cells have abundant rhabdoid to spindled eosinophilic cytoplasm, large eccentric nuclei with prominent nucleoli (Arrow). Present in the background is abundant mixed inflammatory infiltration consisting of lymphocytes, plasma cells, histiocytes, multinucleated giant cells and neutrophils (Arrow). Normal entrapped tubules are highlighted (stars).

Figure 2.

Immunohistochemical staining of biopsy samples obtained from renal neoplasm after renal tumor inoculation. A) The neoplastic cells show positive nuclear PAX8 supporting renal tubular origin. B) Strong diffuse positive nuclear staining for the tumor suppressor gene p53, is supportive of malignant transformation. C) Focal positivity of CK7 suggests epithelial origin (also seen benign tubules highlighted in green). D) The neoplastic cells showed frequent Ki-67 nuclear expression as seen in proliferating cells.

Immunohistochemically the neoplastic cells showed positive expression with cytokeratin AE1/AE3, cytokeratin 7, vimentin and PAX8 supporting a renal tubular origin and epithelioid differentiation. In addition, these cells showed diffuse strong reactivity for p53, moderate Ki-67 and cleaved caspase 3 (20–30%) expression, but lacked carbonic anhydrase IX (CAIX), CD117 (c-Kit), racemase, cathepsin K, TFE3, TFEB, p63, CD10, and PDL-1expression (Table 1).

Table 1.

Immunohistochemistry results and comparison to clinical sarcomatoid (sRCC) or rhabdoid (rRCC) tumors.

Marker	Result	Description
CD10	Negative	Normal renal proximal convoluted tubules and some renal neoplasms
CD31	Positive (endothelial cells)	Endothelial cells
CD68	Positive (macrophages)	Histiocytes / monocytes
CD117 (c-Kit)	Negative	Normal renal distal convoluted tubules, mast cells, some renal neoplasms
Carbonic anhydrase IX	Negative	Hypoxia marker, clear cell renal cell carcinoma
Caspase 3	Positive (20–30%)	Apoptotic cells
Cathepsin K	Negative	Some renal neoplasms
AE1/AE3	Positive (focal positive)	Epithelial cells
Cytokeratin 7	Positive (focal positive)	Epithelial cells, some renal neoplasms
Ki-67	Positive (25–30%)	Proliferating cells
p53	Positive	Tumor suppressor gene, > 5% expression associated with malignant neoplasms
p63	Negative	Some epithelial cells, urothelial carcinoma
PAX8	Positive	All renal tubular cells, all renal cell carcinomas
PDL-1	Negative	Presence of immune checkpoint protein
Racemase	Negative	Epithelial cells of renal proximal tubules
TFE3	Negative	Transcription factor expressed in MiT family translocation RCC
TFEB	Negative	Transcription factor expressed in MiT family translocation RCC
Vimentin	Positive	Mesenchymal cells, some RCC

Discussion

RCC arises from nephric epithelial cells and is a widely variable disease, a large percent of tumors are associated with a loss or mutation of the von Hippel Lindau (VHL) gene in combination with other mutations.^21,22 The ideal model for cancer research would be located in a large animal that possesses similar anatomy and physiology to humans, be site specific, located in the proper organ of study, and contains histologic, genetic, molecular, and immunogenic similarities that accurately replicate the human cancer. However, the vast majority of animal models of RCC are small animal models, typically in mice, rats, and rabbits, secondary to cost and ease in the creation of generic clones.²³ Limited animal models exist for research into RCC and currently no model that accurately mimics s/rRCC is available.

Mice are the most common animal model employed in cancer research with the manipulation of relevant onco- and tumor suppressor genes promoting the in vivo development of RCC.²⁴ In immunodeficient mice, xenograft models have been made from either mouse cell-lines or patient-derived tissue, these tumors retain histology and genetic signatures of the original cell line, thus most accurately mimic human RCC. However, given the importance of checkpoint inhibitor drugs in RCC, the lack of an immune system in these animals limits the accuracy of data obtained and its translation into clinical treatment. Additionally, most xenografts currently employed in research studies are not orthotopic but subcutaneous (s.c.), intraperitoneal (i.p.), or intramuscular (i.m.) due the technical challenges of inoculating the tumor in the mouse kidney.²⁵

The Balb/c mouse derived syngeneic renal adenocarcinoma model (RENCA) can procedure xenograft tumors in an immunocompetent mouse, with tumors that demonstrate a high immune-infiltration similar to clinical tumors.^26,27 However, the immune system and its response between mice and humans is dissimilar, especially compared to humans and pigs. RENCA tumors are mouse not human derived, thus the genetic profile of the tumors is dissimilar to human RCC and have differences in growth rates compared to human RCC. RENCA is currently used to study s/rRCC, although these tumors are not a direct representation of s/rRCC with significant histologic differences.

More recently, genetically engineered mouse models (GEM) with Cre driven gene deletion during early embryogenesis have been created in an attempt to replicate common genetic changes displayed in human tumors and are used to evaluate the origins and progression of disease in addition to how these tumors respond to combination therapy. Some GEM models develop sporadic tumors de novo, supporting investigation in tumorigenesis and progression. Specifically, GEM models have been developed with deletions of both VHL and Pbrm1, the two most commonly mutated genes in RCC which develops spontaneous tumors that display clear cytoplasm, hyperactive mTORC1 signalling, downregulation of critical oxidative phosphorylation genes, and HIF-1α accumulation.²⁸ Deletion of VHL and BRCA1-associated protein-1 created high grade clear cell tumors which were positive for CAIX. A triple mutation of VHL, Trp53, and Rb1 develops site specific tumors with necrosis, tumor haemorrhaging, and nuclear accumulation of HIF-1α and HIF-2α.²⁹ Species-specific genetic differences, such as the chromosomal location of important oncogenes and gene deletion, as opposed to mutation, also limit investigations of interactions conferred by mutated as opposed to absent proteins and sequential loss of function. It is also unknown how genetic deletions affect the immune response compared to mutations.^30,31 Mouse GEM models remain unable to precisely represent the genetic and pathological phenotypes identified in human RCC, and more specifically do not accurately mimic s/rRCC or display associated mesenchymal features, while our tumors exhibited histologic features of sarcoma, including dense spindle-like cells and cellular atypia.

While mouse models are reproducible and economical, many limitations exist concerning their use related to physiologic, genetic, and immune differences. Genetic similarities between humans and mice are only 80% with differences in gene families, duplications, and gene regulation which can have a dramatic impact on the evaluation of therapeutics.³² The translatability of the data concerning drug toxicity, sensitivity, and efficacy obtained from mouse models, particularly xenograft models is also variable and poorly correlates with human results.^33–35 Fewer than 10% of drugs tested in mice translate successfully from animals into Phase 1 clinical cancer trials.¹⁵ The small size of mice also constrains the testing of novel procedural techniques, such as intravascular drug delivery of tumor specific theranostic agents, use and validation of clinical devices, and combination techniques such as local personalised ultra-fractionated stereotactic adaptive radiotherapy (PULSAR) external beam radiation with chemotherapy. These are all widely employed in clinical practice and their importance continues to grow in the future of oncologic treatment.

Our study evaluated a transgenic GEM porcine pig, which contains Cre-inducible KRASG12D and TP53R167H mutations, as a method to develop a site and cell specific tumors that can function as a large animal model for RCC model. The image guided tumor inoculation of the oncopig in the kidney procedure was safe, simple, site-specific, reproducible, and resulted in rapidly growing renal tumors that can be identified two to three weeks post-inoculation. Although large animals are expensive to use in research; the similarities of physiology, size, anatomic and vascular anatomy, and immune system between swine and humans, produce an ideal target for studies evaluating interventional therapies such as percutaneous thermal ablation, external beam radiology treatments, as well as surgical techniques.

Evaluating the previous experience from other groups who used this oncopig model to induce tumors within the liver, pancreas, and lung, for our study, we decided to investigate only the in-situ approaches of tumor inoculation. This in vivo technique uses a tissue biopsy sample obtained from the organ of interest that is incubated in AdCre virus outside the body. Subsequently, the incubated mixture is returned to the original organ percutaneously. For renal tumors, inoculation occurred at the expected location of the corticomedullary junction using ultrasound via a coaxial needle. Our success rate of 88% was comparable to those obtained by other groups using a similar technique in the other organs of the oncopig (92% liver, 86% pancreas, 83% lung).^18–20 The cell line method is an alternative approach to inoculate an organ with a tumor. When employing the cell line method, virus alone is directly injected percutaneously or endovascularly into the organ in question and has been previously attempted, however, the success rates were significantly lower in other organs than the in-situ. In the kidney, the consideration was made for the intra-arterial infusion of the virus or the incubated renal tissue, virus, and gelfoam slurry mixture, however given that the previously reported success rate was negligible, and we desired to have a cortical tumor, not central or medullary tumors we did not investigate this protocol. This technique can potentially be researched in the future to simulate a renal tumor with vascular invasion.

s/rRCC is not a distinct histologic or morphogenetic subtype of RCC but is a specific dedifferentiation of both the mesenchymal and epithelial cells that can occur within any type of RCC. The presence of these changes are associated with more aggressive features, reports have indicated an increased percent of sarcomatoid dedifferentiation on histology is associated with an overall worse prognosis for patients.³⁶ In patients with RCC without sarcomatoid dedifferentiation surgical resection of the localised tumor is curative, however in 80% of patients with localized s/rRCC, recurrence occurrences 5–26 months after surgery.³⁷ The appearance of s/rRCC is disproportionately identified in metastatic RCC. Systemic treatments for s/rRCC have also proven to be less effective in killing this disease.^38,39 Having a model that accurately mimics the tumor biology of s/rRCC would be vital in the preclinical evaluation of novel therapies.

Genetic evaluation of s/rSCC has shown an increase in TP53 mutations within their sarcomatoid component.⁴⁰ Next generation sequencing of sRCC verified these results and also identified mutations in the PTEN gene, increased expression of two other cancer driver genes, BAP1and ARID1A, and amplification of JAK2 were also reported.^41,42 The oncopigs have an inducible mutation of the TP53R167H gene, this is the porcine equivalent of the human TP53R175H mutation, mirroring some genetic changes seen in s/rRCC. Additional amplifications of the PDL1 and PDL2 genes were also seen in s/rRCC and were correlated with IHC- based programmed cell death 1 ligand 1 (PDL1) expression.^43,44 However, IHC evaluation of our tumor demonstrated minimal to no expression of PDL1, this is disappointing, and we believe is due to the extensive intrinsic inflammatory reaction this tumor model undergoes. Additionally, the mutations activated in the oncopig are not related to changes in the PDL1 genes. As previously reported the tumor also displays an epithelial origin with cytokeratin AE1/AE3, cytokeratin 7, vimentin and PAX8 positivity supporting a renal tubular origin.

A porcine renal tumor model has previously been reported which created orthotropic pseudotumors in the kidney by injected various substances, such as gelatin and psyllium into a porcine kidney, creating a mass in the kidney that could be used to practice surgical techniques but did not display any specific neoplastic qualities of an RCC.⁴⁵

Several limitations can be identified in the oncopig model, first and foremost, this model is significantly more expensive than a small animal model and requires a much greater input from the veterinary staff at your institution to complete experiments. However, the high initial cost may be counteracted by improved data that can provide information which has greater clinically relevance from using our renal tumor model that can more quickly be translated into humans. Another limitation is that the tumor is not an exact reproduction of human s/rRCC, but this is similar to the currently available renal xenograft and GEM models used in preclinical RCC research today as no exact replica for s/rRCC is available for research at this time. The inoculation of tumors results in a large amount of associated inﬂammation which may be dissimilar to the immune reactions exhibited in human RCC. The oncopig tumors also grow rapidly, with masses observed between 14–20 days, this may disrupt the development of the extracellular matrix and neovascularity that is seen in human tumors which may take a longer time to mature and develop, potentially making our tumors dissimilar to clinical RCC. The natural progression of the tumor over a prolonged period of time was also not evaluated, as data was only obtained at early time points after inoculation, within weeks. Our study was unable to characterise renal tumor perfusion with advanced angiography such as CTA or cone-beam CT, this prevents us from a direct comparison of the tumor vascularity in the oncopig model to humans. However, previous tumors like those in the liver developed vascularity similar to primary or metastatic hepatic tumors, we hope to obtain this characterization in future experiments.

Conclusion

The creation of site-specific tumors in the kidney was shown to be safe and feasible with high rates of success after the image guided inoculation of the renal corticomedullary region. Poorly differentiated neoplasms grew rapidly with pathology supporting a renal tubular origin with presences of additional features suggesting sarcomatoid (sRCC) or rhabdoid (rRCC) changes. The presence of tumors within a large animal, which has comparable size, genetic, anatomic, and physiologic features to humans can be used to successfully to evaluate novel treatment in preclinical studies particularly interventional and surgical therapies.

Footnotes

Acknowledgements

The authors have no acknowledgments.

ORCID iDs

Samuel L Rice

Payal Kapur

Luis De Las Casas

Ethical considerations

This study was performed after approval by our Institutional Animal Care and Use Committee (IACUC approval number 103088).

Author contributions

All authors: conception and design. data collection and analysis: Samuel L Rice, Mhd Wisam Alnablsi, Payal Kapur, Luis De Las Casas. All authors: read and approved manuscript. All have access to data

Funding

Funding for this research was provided by an investigator-initiated grant from Johnson and Johnson NW-2020-04 entitled “In Vivo Evaluation of Ablation Zones With and Without Prior Intra-Arterial Embolisation in an Oncopig Renal Tumor Model” and NW-2020-03 entitled “In Vivo Evaluation of Ablation Zones With and Without Prior Intra-Arterial Embolization in an Oncopig Renal Tumor Model”.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Development and characterisation of a large animal transgenic tumor model for sarcomatoid renal cell carcinoma

Abstract

Background

Objective

Materials

Results

Conclusion

Keywords

Introduction

Methods and materials

Tumor induction (in situ method)

Imaging

Pathology

Results

Inoculation success

Renal tumor pathology

Discussion

Conclusion

Footnotes

Acknowledgements

ORCID iDs

Ethical considerations

Author contributions

Funding

Declaration of conflicting interests

Data availability

References