The use of donor leukocyte infusions (DLI) following allogeneic HCT to both augment anti-tumor responses and enhance immune cell engraftment has expanded dramatically since its introduction 30 years ago (54). DLIs utilize donor peripheral blood leukocytes collected via apheresis, a process in which blood components are separated via density gradient and lymphocytes and monocytes are harvested, while granulocytes are returned to the patient. DLI following HCT in the setting of hematological malignancy has several indications, including as a prophylactic therapy for patients with a high risk of relapse, treatment of PTLD and viral infections and as a rescue therapy for those with graft failure. However, GVHD remains one of the most feared side effects of allogeneic BMT. Important studies by Sachs' group demonstrated the value of swine as a cell therapy model to optimize and harness the anti-leukemia effects of allogeneic HCT while avoiding GvHD. These studies in swine exploited a novel HCT approach first shown in mice in which the establishment of mixed chimerism across MHC barriers promoted immune tolerance, thus preventing GvHD (55, 56). This study and other similar studies in non-human primates laid the foundation for Sachs' pioneering study in humans using allogeneic BMT to simultaneously treat multiple myeloma and induce immune tolerance to MHC mismatched kidney allografts (57). Using the swine model described above, we attempted to leverage the alloreactive properties of DLIs to enhance donor chimerism, thereby maintaining or preventing graft loss, while at the same time avoiding GvHD (42). In our study of a total of 33 clinically dosed DLIs infused to immune tolerant swine chimeras, 21 failed to induce conversion to full donor hematopoietic chimerism or cause GvHD, demonstrating that our reduced intensity conditioning regimen for HCT promotes mixed chimerism and an immune tolerant state that is strongly resistant to DLI and GvHD. In several animals, we were able to demonstrate that DLI mediated GvH reactivity could be overcome by significantly increasing the DLI dose, removing chimeric host peripheral blood cell populations (thought to be regulatory T cells) through extensive leukapheresis of the recipient immediately prior to DLI, or delivering lymphocytes fully mismatched to host MHC, but not to donor MHC. However, conversion to full donor chimerism in these scenarios was often associated with severe GvHD, highlighting the importance of mixed chimerism for maintaining immune tolerance. More refined DLIs with selected effector populations are currently being developed and swine will play an important role in determining their efficacy.

### γδT Cells Infusions

γδT cells are a conserved subset of T cells with a distinct surface receptor and which mediate innate immune responses and promote immune surveillance (58). The abundance of γδT cells in humans ranges from 1 to 20% in the peripheral blood and constitutes the major cell population in skin and mucosa (59). Swine γδT cells have been previously characterized and can be tracked in the peripheral blood and tissues using the monoclonal antibody PPT27 (58, 59). γδT cells play a known role in the anti-tumor response and can therefore potentially be used as a potent cellular therapeutic in the context of BMT. However, infusion of donor type γδT cells in a murine model of acute GVHD (aGHVD) mice increased the severity of aGVHD while the absence of host type γδT cells was associated with reduced antigen presenting cell (APC) activation and aGVHD in an MHC-mismatched model (59). By contrast, aGVHD severity was not altered in a MHC-matched, minor antigen (miHA) disparate model of HCT (58, 60). Studying the role of γδT cells in the setting of MHC disparity using a large animal model may prove useful amidst growing evidence of the immune modulatory effects of these cells.

γδT cells provide potent anti-tumor responses to both solid and hematopoietic malignancies, including lymphoma and multiple myeloma (61). As opposed to αβT cells, γδT cells are not MHC restricted when it comes to antigen recognition and do not require APCs for processing immunogenic peptides, allowing them to quickly reactivate during an immune response. Interestingly γδT cells can be activated and expanded in vivo through the use of bisphosphonates, which inhibit farnesyl pyrophosphate synthase (62). Taken together, the unique immune properties of γδT cells, as well as their ability to be activated in vivo using conventional drugs, make them an attractive option for experimental use in swine models of HCT. Thus far, there have been limited studies utilizing γδT cells in preclinical models or clinical settings. Using a large animal preclinical model such as swine, where γδT cells have been well-characterized, could allow for optimization of important parameters including dosing, route (systemic vs. intratumoral), kinetics, and ex-vivo manipulation. More importantly, safety studies in an outbred swine will help discern the conflicting γδ T cell GVHD murine studies and facilitate the expanded use of γδT cells in clinical settings.

### NK Cell Therapies

NK cells are a type of innate lymphoid cell that mount allogeneic immune responses in a non-MHC restricted manner. NK cells distinguish "self " vs. "non-self " through interactions between the killer inhibitor receptor (KIR) expressed on their surface and HLA class I expressed on the surface of host tissues. Recognition of "self " HLA class I by the KIR results in an inhibitory signal, while the absence of HLA class I expression stimulates NK cell activation. NK cells play a pivotal role in the anti-tumor response in cancer cells that down-regulate HLA expression to escape recognition by T cells.

Clinically, NK cells provide a powerful anti-leukemia effect in the setting of allogeneic HCT. For the treatment of acute myelogenous leukemia (AML), donor NK cell alloreactivity from KIR mismatched donors displayed an anti-leukemia effect as part of T cell depleted grafts, while simultaneously providing protection against GVHD (63). Based on these anti-leukemic effects in the absence of GVHD, adoptive NK cell therapy has also been studied as an alternative to unmanipulated DLI for leukemic relapse. NK-DLI was demonstrated to be both a feasible and safe option in a study of 30 patients receiving nonmyeloblative allogeneic stem cell transfer (SCT). CD56+ selected NK cells were given as an NK-DLI 8 weeks after initial transplant. Patients tolerated the DLI well without significant GVHD (64). In the non-transplant setting, administration of KIR mismatched NK cells in 10 pediatric patients with AML who had achieved complete remission following chemotherapy resulted in transient engraftment and excellent two year overall survival (100%) (65). Studies are also underway to evaluate the potential for activated NK cell therapy in the setting of refractory lymphoma (66).

The recent development of the SCID pig provides an interesting avenue for studying the role of NK cells in both allogeneic and xenogeneic anti-tumor responses in a large animal model (67). Similarly to human NK cells, swine NK cells can be identified by expression of CD3−CD16+CD56+ surface markers (68). Powell et al. demonstrated that the number of NK cells in SCID pigs is approximately eight times higher than in non-SCID pigs and that these NK cells are intrinsically functional, as demonstrated by their ability to be activated in vitro and lyse tumor cells at the same rate as NK cells from non-SCID pigs (69). In the absence of circulating T and B cells, methods to activate and harness NK cell immune responses can be specifically evaluated in SCID pigs. For example, as IL-2 can stimulate NK cells against ovarian cancer in a murine model (70), further evaluation of IL-2 and other therapies in the SCID pig may further delineate the clinical relevance of NK cells in anti-leukemia and anti-lymphoma immune responses.

### Chimeric Antigen Receptor (CAR) Cells

CARs are genetically engineered receptors that reprogram T cells to target specific cell surface antigens without the need for MHC interaction. CAR T cell therapy has revolutionized cancer research through the re-direction of T cells to target surface receptors expressed by tumor cells, most notably CD19 which is expressed on B cell malignancies (71). CAR T cells were first shown to have anti-tumor responses in mice and then in humans with refractory hematological malignancies (71–73). Currently, groups are designing novel CARs for applications other than cancer, such as autoimmunity and infectious disease (74). As a result, there is an increased need to test the safety of many of these. Indeed, fatal side-effects have been observed in clinical trials, which argues for the need for improved safety testing prior to clinical application, ideally in large animal models (75).

Given the availability of swine models of B cell lymphomas and myeloid leukemias and the identification of the SCID pig, swine may provide an ideal large animal model for the testing of CAR therapies. Though further refinement of these models is necessary, their use should be encouraged for safety assessments in pre-clinical studies. Because CARs rely on the insertion of gene sequences coding for a monoclonal antibody (with a given antigen specificity) as part of the receptor, a potential limitation of swine models is that the CAR may not recognize the target antigen on swine cells. Though this is a potential limitation, for some constructs with conserved antigens, CARs can be very informative for "off-target effects" (76). Swine may be particularly valuable for assessing the relative kinetics and persistence of individual CARs, as it was recently shown that 4-1BB CARs are longer lived when compared to CD28 CARs in humans (77). The potential use of SCID pigs engrafted with human leukemias/lymphomas to assess cytotoxicity/clearance, dosing, imaging, CAR surveillance and different systemic/local delivery methods may be revolutionary in a field that has been mostly limited to murine models. Furthermore, swine could also be used to test the efficacy of anti- PTLD therapies by either CARs directed to PLHV-1 in the MGH swine or to EBV CARs in the case of humanized swine. The possibilities are endless depending on the approach and research questions asked, and highlight the potential role of swine as a critical player in the pre-clinical space of cellular immunotherapies.

### Immune Checkpoint Blockade

Activation of the host immune system against invading tumor cells has long been the goal of cancer therapeutics. A major breakthrough in this endeavor was the discovery of immune checkpoint proteins, which serve to downregulate the immune response. The first immune checkpoint protein to be wellcharacterized was cytotoxic T-lymphocyte-associated protein 4 (CTLA4), a receptor found on the surface of regulatory T cells and activated T cells. When CTLA4 is bound to its ligands, CD80 and CD86, on the surface of APCs, it provides an inhibitory signal to the T cell. However, the activation molecule CD28 also binds to CD80/CD86, albeit with a reduced affinity as compared to CTLA4. Thus, in the setting of solid organ transplantation where the goal is to suppress the host immune response to the allogeneic graft, it was hypothesized that CTLA-4 would block CD28 interactions with CD80/86, thereby preventing T cell activation. In support, Belatacept, a novel CTLA4 Ig fusion protein, is an effective form of immunosuppression in organ transplantation in both large animal models and humans (78, 79). Naturally, in the setting of cancer, blocking the interaction between CTLA4 and CD80/86 would serve to activate circulating T cells and theoretically fight off invading tumor cells. Ipilumamb, a monoclonal antibody directed against CTLA-4, was first approved by the FDA in 2011 for the treatment of metastatic melanoma and has provided excellent results (80).

The porcine version of CTLA-4 (pCTLA4) exists in several forms and can suppress human CD4+ T cell responses costimulated by porcine B7. Utilizing a novel diptheria toxin (DT) based recombinant pCTLA4 fusion toxin, Peraino et al. demonstrated effective binding to CD80 expressing porcine cells and subsequent inhibition of protein synthesis in those cells. Follow up studies in mice inoculated with a CD80+ porcine lymphoma cell line showed that mice injected with the DT based pCTLA4 fusion toxin experienced prolonged survival compared to untreated mice. It remains to be seen whether the use of pCTLA-IT has similar effects in swine as what has been observed in murine studies where blocking or removing (genetically) host APCs diminished GVHD by limiting the direct activation of alloreactive T cells (81). In summary, the demonstrated ability of a DT based pCTLA4 to inhibit growth of porcine lymphoma cells provides a foundation for future work in targeting CTLA4 in large animal models of lymphohematopoietic malignancies.

### CONCLUSIONS

Cancer research is currently being revolutionized by the development of novel cellular and genetic therapies. Historically, these strategies required testing in small animals due to the absence of reliable large animal cancer models. However, recent advancements in swine including the development of immortalized myeloid and lymphoma cell lines from inbred MHC characterized swine, the accessibility of genetically engineered oncogenic swine (known as oncopigs and addressed in a companion review in this series), and the ability of engrafting human tumors in the SCID

### REFERENCES

pig, highlight swine as an ideal model for large animal tumor studies.

### AUTHOR CONTRIBUTIONS

RD-S: Lead the topic. CH: Provided figures and edited the paper with modifications. RD-S and AM: Wrote the manuscript.

### FUNDING

NIH- SERCA award- 1K01RR024466. University of Pennsylvania Internal Funds.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Duran-Struuck, Huang and Matar. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Translating Human Cancer Sequences Into Personalized Porcine Cancer Models

Chunlong Xu1 , Sen Wu1 , Lawrence B. Schook 2,3,4 and Kyle M. Schachtschneider 2,4,5 \*

*1 State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China, 2 Department of Radiology, University of Illinois at Chicago, Chicago, IL, United States, 3 Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL, United States, 4 National Center for Supercomputing Applications, University of Illinois at Urbana-Champaign, Urbana, IL, United States, 5 Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL, United States*

The global incidence of cancer is rapidly rising, and despite an improved understanding of cancer molecular biology, immune landscapes, and advancements in cytotoxic, biologic, and immunologic anti-cancer therapeutics, cancer remains a leading cause of death worldwide. Cancer is caused by the accumulation of a series of gene mutations called driver mutations that confer selective growth advantages to tumor cells. As cancer therapies move toward personalized medicine, predictive modeling of the role driver mutations play in tumorigenesis and therapeutic susceptibility will become essential. The development of next-generation sequencing technology has made the evaluation of mutated genes possible in clinical practice, allowing for identification of driver mutations underlying cancer development in individual patients. This, combined with recent advances in gene editing technologies such as CRISPR-Cas9 enables development of personalized tumor models for prediction of treatment responses for mutational profiles observed clinically. Pigs represent an ideal animal model for development of personalized tumor models due to their similar size, anatomy, physiology, metabolism, immunity, and genetics compared to humans. Such models would support new initiatives in precision medicine, provide approaches to create disease site tumor models with designated spatial and temporal clinical outcomes, and create standardized tumor models analogous to human tumors to enable therapeutic studies. In this review, we discuss the process of utilizing genomic sequencing approaches, gene editing technologies, and transgenic porcine cancer models to develop clinically relevant, personalized large animal cancer models for use in co-clinical trials, ultimately improving treatment stratification and translation of novel therapeutic approaches to clinical practice.

Keywords: personalized cancer models, exome sequencing, gene editing, translational research, clinical needs

### INTRODUCTION

The global incidence of cancer is rapidly rising, and despite an improved understanding of cancer molecular biology, immune landscapes, and advancements in cytotoxic, biologic, and immunologic anti-cancer therapeutics, cancer remains a leading cause of death worldwide. The 14.1 million new cancer cases diagnosed in 2012 are expected to dramatically increase over the next decade to 19.3 million annual cases by 2025 (1). Cancer is caused by the accumulation of a series of gene mutations

### Edited by:

*Michael Breitenbach, University of Salzburg, Austria*

#### Reviewed by:

*Maja Cemazar, Institute of Oncology Ljubljana, Slovenia Daniele Vergara, University of Salento, Italy*

> \*Correspondence: *Kyle M. Schachtschneider<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Molecular and Cellular Oncology, a section of the journal Frontiers in Oncology*

Received: *17 October 2018* Accepted: *04 February 2019* Published: *25 February 2019*

#### Citation:

*Xu C, Wu S, Schook LB and Schachtschneider KM (2019) Translating Human Cancer Sequences Into Personalized Porcine Cancer Models. Front. Oncol. 9:105. doi: 10.3389/fonc.2019.00105*

**83**

called driver mutations that confer selective growth advantages to tumor cells (2). The development of next-generation sequencing technology has made the evaluation of mutated genes possible in clinical practice, allowing for identification of driver mutations underlying cancer development in individual patients. This, combined with frequency and function-based methods allows for distinguishing potential driver mutations from passenger mutations that have no effect on tumorigenesis. These advances have provided unique insights into the wide variety of genetic alterations present in an individual patient's tumor, and have spurred interest in utilizing this information to inform treatment stratification. However, translation of this genomic information into improved therapeutic approaches has not been successful for the majority of cancer patients. Therefore, as cancer therapies move toward personalized medicine, improved modeling capabilities for predicting the role driver mutations play in therapeutic susceptibility are required to address this unmet clinical need.

Recent advances in gene editing technologies such as CRISPR-Cas9 have enabled development of tumor models with specific genetic driver mutations. When applied to murine cancer models, these targeted genetic alterations have provided key insights into key mutational events promoting tumor progression and altered response to therapy (3, 4). However, many drugs showing promise in murine studies fail to translate into successful clinical trials (5), highlighting the need for improved models to better translate therapeutic efficacy, optimal dosing, and ideal combination therapies to clinical practice. Pigs represent an ideal animal model for development of genetically defined tumor models due to their similar anatomy, physiology, metabolism, immunology, genetics, and epigenetics compared to humans (6–14). In addition, their similar size permits utilization of the same instrumentation and technical maneuvers used in humans and optimized by clinicians, facilitating rapid clinical translation.

As cancer therapies move toward personalized medicine, predictive modeling of the role driver mutations play in tumorigenesis and therapeutic susceptibility will be essential. Combining porcine cancer models and gene editing technology would allow for development of clinically relevant personalized tumor models for prediction of treatment responses for mutational profiles observed clinically. Such models would support new initiatives in precision medicine, provide approaches to create disease site tumor models with designated spatial and temporal clinical outcomes, and create standardized tumor models analogous to human tumors to enable therapeutic studies. In this review, we discuss the process of utilizing genomic sequencing approaches, gene editing technologies, and transgenic porcine cancer models to develop clinically relevant, personalized large animal cancer models for use in coclinical trials, ultimately improving treatment stratification, and translation of novel therapeutic approaches to clinical practice.

### INFLUENCE OF DRIVER MUTATIONS ON TREATMENT RESPONSE

Cancer is caused by the accumulation of a series of gene mutations called driver mutations that confer selective growth advantages to tumor cells (2). The development of nextgeneration sequencing technology has made it possible to evaluate mutated genes in tumor cells. This, combined with frequency and function-based methods allows for distinguishing potential driver mutations from passenger mutations that have no effect on tumorigenesis. Although our understanding of the role various mutations play in driving tumorigenesis is incomplete, it is clear that genetic mutations are found in all cancers, some of which have been associated with biological characteristics of cancer (15). While all tumors result from genetic mutations, each tumor type develops mutations at different rates. In the instance of HCC, it is estimated that a single tumor contains 30–40 mutations on average, 5–8 of which are likely driver mutations (16, 17). Some of these driver mutations can have profound effects on tumor biology, having significant implications regarding diagnostics, prognostics, and therapeutic responses. For example, mutation of the tumor suppressor gene TP53 is associated with poor prognosis and doxorubicin resistance in HCC (2, 18–20), while RAS activation is associated with resistance to sorafenib (2). Other examples include KRAS mutations associated with epidermal growth factor receptor antibody resistance in colorectal cancer (15), and BRAFV600E mutations associated with positive response to vemurafenib in melanoma patients (21). As genomic analyses of clinical cancer samples continues to increase, and databases such as The Cancer Genome Atlas (TCGA) continue to grow, so does our understanding of the mutations that impact treatment recommendations. However, despite the knowledge that driver mutational profiles can have significant impacts on treatment responses, tumor genomic information is not routinely used when considering treatment strategies for the vast majority of cancer types. The lack of translation into actionable therapeutic modalities highlights the need to develop novel platforms to rapidly analyze and predict therapeutic responses for patients based on their driver mutational profiles.

### CO-CLINICAL TRIAL CONCEPT

With increased interest in testing targeted therapeutics based on driver mutational profiles in cancer patients comes a significant decrease in the number of relevant patients available for enrolment in appropriate clinical trials, significantly reducing the number of new targeted and combination therapies that can be tested. One of the new ways investigators are attempting to address this issue is through the use of co-clinical trials. Co-clinical trials are defined by the National Cancer Institute (NCI) as parallel or sequential trials of combination therapy in patients and in mouse and human-in-mouse models of appropriate genotypes to represent the patients. Utilization of mouse models that mimic the genetics of human disease in parallel to early phase human clinical trials can assist in treatment stratification by identifying patient populations most likely to benefit from treatments based on their genetic makeup. These so called "mouse hospitals" enable testing of drugs in murine models representative of multiple cancer subtypes while minimizing the cost, time, and number of human patients required (4). Co-clinical trial approaches using genetically engineered mouse models (GEMMs) have shown promise for screening therapeutics and identifying patient populations that would benefit from specific treatments (4). However, GEMMs have several drawbacks that limit the translatability of results to clinical practice. The metabolic rate of mice is substantially higher than in humans (22), and vast differences in drug metabolism and xenobiotic receptors make rodents poor models of toxicity, sensitivity, and efficacy when used in preclinical drug studies (23). The ability to establish toxicity and drug sensitivity in animal models is immensely important, as <8% of cancer drugs translate successfully from animal model testing into Phase I clinical trials (24). In addition, their small size prohibits the utilization and testing of the same tools and techniques employed in clinical practice. This is particularly important given the recent expansion of targeted locoregional ablative and arterial therapeutic strategies that reduce systemic toxicities and increase tumor drug delivery. This, combined with the fact that the genetic events required for mouse tumorigenesis differs from humans (25), highlights the need for development of improved animal models to facilitate translation of targeted and personalized therapeutic strategies to clinical practice.

### Argument for Porcine Cancer Models

Given the limitations of currently available murine and other small animal cancer models, there is a pressing need to incorporate large animal cancer models into preclinical and co-clinical therapeutic testing approaches. Pigs represent an ideal platform for development of genetically defined large animal cancer models due to their similarities with humans in size, anatomy, physiology, metabolism, genetics, epigenetics, and immunology (6–14). The life cycle of pigs also allows for development, characterization, treatment, and follow-up in a clinically relevant timeframe (26). The availability of many outbred porcine lines, high homology between the pig and human genome (27, 28), and conservation of epigenetic regulatory patterns (13) highlights the relevance of genetically defined porcine cancer models and their ability to mimic the genetic variation observed in patient populations. Pigs are also ideal models for investigation of chemotherapeutic toxicity, as the animal's basal metabolic rate and xenosensor pregnane X receptor—which is responsible for the metabolism of half of all prescriptions drugs (29)—are also very similar to humans (30, 31). Finally, their similar size allows for utilization of the same tools and techniques used in clinical practice. This is particularly important for cancers where systemic chemotherapeutic administration offers only marginal survival benefit with poor quality of life, as procedural approaches using locoregional therapeutic approaches are potentially curative therapeutic options that require further preclinical testing, but cannot be tested using similar tools in smaller animal models.

Until recently the only porcine cancer models available were spontaneous or chemically induced models (32–34). However, the sequencing of the pig genome in combination with the recent advances in targeted genome editing approaches such as CRISPR-Cas9 has allowed for development of genetically defined porcine cancer models. To date a number of genetically defined porcine cancer models capable of mimicking histological and transcriptional hallmarks of human cancer, as well as responses to cancer drug therapies have been developed. These include the Oncopig Cancer Model—a transgenic pig model that recapitulates human cancer through induced expression of heterozygous KRASG12D and TP53R167H driver mutations which has been utilized to develop HCC (35), pancreatic cancer (36), and soft-tissue sarcomas (37, 38), and a heterozygous TP53 knockout model of spontaneous osteosarcomas (39). As genetically defined porcine cancer models continue to be developed, their use in co-clinical trial formats could provide improved prediction of patient populations that would benefit from specific treatments, improving translation of novel, targeted, and combination therapeutic strategies from preclinical murine studies to clinical practice. As our understanding of driver mutational profiles commonly observed in clinical practice continues to expand thanks to the increased use of genomic sequencing in clinical research, this information can serve as a basis for generation of additional porcine cancer models using CRISPR and somatic cell nuclear transfer (SCNT) technologies.

While there are a number of benefits associated with the use of genetically defined porcine cancer models in co-clinical trial settings, these models are not without limitation. Drawbacks of using porcine models as opposed to murine models include increased housing and husbandry requirements due to their increased size and lifespan. This limitations also limits the ability to develop, breed, and distribute multiple strains of porcine cancer models harboring different driver mutations as is currently done for murine models. In addition, specialized equipment and experience are required to ensure safe and ethical handling and use of pigs for testing experimental treatments. These animals are also raised in controlled environments that do not mimic the environmental conditions human patients are exposed to—although as this limitation is shared with murine and other cancer models, a detailed discussion of the environmental factors impacting tumor biology and treatment response is outside the scope of this review. Finally, the costs associated with development, maintenance, and utilization of porcine cancer models in co-clinical trials is significantly higher than murine models, although their use would come at a reduced cost compared to those associated with human clinical trial participants.

### TREATMENT STRATIFICATION UTILIZING PERSONALIZED PORCINE CANCER MODELS

Our increased understanding of the unique genetic makeup of each patient's tumor has shed light on the fact that individual cancer varieties exist, and therefore therapies need to be optimized and adjusted to effectively treat individual patients. This optimization requires the use of preclinical cancer models representative of the driver mutational profiles of individual patients. While current co-clinical trials seek to utilize genetically defined murine cancer models in combination with human cancer patients to evaluate treatment response for patient populations, personalized porcine cancer models could transform precision medicine by providing a means to

significantly improve the predictability of safety and efficacy of therapeutic drugs, devices, and procedures in co-clinical trial settings (**Figure 1**). Below we outline the process for developing genetically defined, personalized porcine cancer models, using the Oncopig HCC model as an example.

The first step in developing personalized porcine tumor models consists of identification of the driver mutational profile for which the treatment in question is most likely to be effective against. For targeted therapeutics, this can be done using preclinical murine models prior to proceeding with co-clinical trials utilizing personalized porcine tumor models. For repurposed compounds already approved for other cancer types, this would require knowledge of the driver mutational profiles of responding an non-responding patients. This requires performance of biopsy collection, followed by DNA extraction and genomic sequencing—for example through whole genome or whole-exome sequencing—to identify the driver mutations present. Sequencing of a control sample, such as blood, is also required to assist in distinguishing between germline and somatic mutations. Utilizing genome editing approaches such as CRISPR-Cas9, driver mutations associated with improved outcomes can be introduced into the porcine HCC cells in vitro. Following screening to identify cells containing the desired driver mutational profile, HCC cells are propagated for autologous injection, resulting in development of pigs bearing HCC tumors with driver mutational profiles representative of the patients of interest. Utilizing this approach, a cohort of personalized porcine cancer models can be developed in a timely fashion and utilized in co-clinical trials, significantly reducing the costs and accrual time associated with clinical trials. This approach would also provide significant benefits over murine co-clinical trials by utilizing a model animal with similar metabolism and size to humans, allowing for the same tools and techniques to be employed in both human and porcine subjects.

While the above example describes utilization of the Oncopig Cancer Model to develop personalized HCC tumors, this approach is not limited to the Oncopig and can be adjusted to facilitate development of personalized tumors for a wide variety of cancer types. However, due to the above mentioned challenges associated with developing, breeding, and disseminating multiple strains of porcine cancer models, it is unlikely that the breadth of porcine cancer models required for co-clinical trials targeting specific driver mutational profiles will ever match the number of commercially available murine models. Therefore, development of various cohorts of genetically defined porcine cancer models for co-clinical trials will likely depend on utilization of CRISPR-Cas9 to induce tumors harboring desired driver mutational profiles in individual wild type or previously produced inducible porcine cancer models. While this approach provides additional challenges compared to utilization of genetically defined murine models, it also allows for rapid development of genetically defined porcine tumor models without the extended time required to develop a pig herd harboring the desired mutational profile. In this regard, pigs harboring tumors representative of multiple driver mutational profiles could be used as their own control to confirm the effects of a given driver mutational profile on treatment response. This approach could also revolutionize personalized medicine by facilitating development of genetically unique, patient specific tumors for performance of therapeutic trials on tumors representative of the genetic profile of individual patient tumors. However, much work is still required to make this approach feasible in a timely and cost efficient manner.

### Accounting for Intratumoral Heterogeneity

One of the challenges faced when developing personalized tumor models is accounting for intratumor heterogeneity, which describes the accumulation of different genetic mutations in tumor cells within a single tumor as tumor cells evolve (40). Knowledge of the genetically diverse cell populations within a tumor can be important for guiding optimal cancer treatment decisions, and therefore the effectiveness of personalized tumor models to predict the optimal treatment strategy may be underappreciated when used to treat heterogeneous tumors. While tumor cells representative of the driver mutations modeled will be killed, the patient may develop a recurrent tumor or not respond at all due to proliferation of resistant tumor cells. These situations highlight the importance and significant challenge associated with performing clinical and co-clinical trials for targeted therapeutics, as well as the challenges of successfully employing them in clinical practice. While modeling heterogeneity represents a significant challenge for animal cancer models, the need to perform gene editing on individual pigs as described above provides an avenue through which tumor heterogeneity can be accounted for using personalized porcine cancer models. Porcine cell lines representative of multiple driver mutational profiles can be developed, mixed, and injected to develop in vivo intratumor heterogeneity representative of the patient population of interest. In this case, therapies will only prove effective if they're capable of eradicating all of the

### REFERENCES

genetically distinct tumor cells present. Another option would consist of development of individual tumors representative of one of the genetically diverse tumor cell populations. Using this approach, treatment strategies can be applied to tumors representative of different driver mutational profiles in isolation, allowing for identification of treatment strategies most effective for each tumor cell population. However, these approaches due not take into account additional challenges associated with modeling tumor heterogeneity, including accurate identification of individual tumor clones, effects of cellular signaling and interactions between tumor cells with differential mutational profiles, and the impact of germline mutations on tumor biology.

## CONCLUSIONS

Advances in sequencing and gene editing technologies have provided significant insights into the impact of driver mutations on treatment responses for a wide range of cancer types; however, translation of this genomic information into improved therapeutic approaches has not been successful for the majority of cancer patients. We present a new personalized porcine cancer model approach leveraging clinical genomic sequence information, gene editing technologies, and transgenic porcine cancer models to develop clinically relevant, personalized large animal cancer models to better predict response to treatment in co-clinical trial settings, ultimately improving treatment stratification and translation of novel therapeutic approaches to clinical practice. Furthermore, as these techniques continue to improve, this approach could revolutionize personalized medicine by facilitating development of genetically defined porcine cancer models representative of individual patients for performance of personalized therapeutic trials.

## AUTHOR CONTRIBUTIONS

CX, SW, LS, and KS conceptualized and wrote the manuscript.

support pigs as a biomedical model. BMC Genomics (2015) 16:743. doi: 10.1186/s12864-015-1938-x

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Xu, Wu, Schook and Schachtschneider. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Exploring the Potential Utility of Pet Dogs With Cancer for Studying Radiation-Induced Immunogenic Cell Death Strategies

### Timothy M. Fan\* and Kimberly A. Selting

Comparative Oncology Research Laboratory, Department of Veterinary Clinical Medicine, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, United States

#### Edited by:

Dhanansayan Shanmuganayagam, University of Wisconsin-Madison, United States

#### Reviewed by:

Jan Theys, Maastricht University, Netherlands Gabriele Multhoff, Technische Universität München, Germany

> \*Correspondence: Timothy M. Fan<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Molecular and Cellular Oncology, a section of the journal Frontiers in Oncology

Received: 06 September 2018 Accepted: 27 December 2018 Published: 15 January 2019

#### Citation:

Fan TM and Selting KA (2019) Exploring the Potential Utility of Pet Dogs With Cancer for Studying Radiation-Induced Immunogenic Cell Death Strategies. Front. Oncol. 8:680. doi: 10.3389/fonc.2018.00680 Radiotherapy serves as a foundational pillar for the therapeutic management of diverse solid tumors through the generation of lethal DNA damage and induction of cell death. While the direct cytotoxic effects of radiation therapy remain a cornerstone for cancer management, in the era of immunooncology there is renewed and focused interest in exploiting the indirect bystander activities of radiation, termed abscopal effects. In radioimmunobiologic terms, abscopal effects describe the radiotherapy-induced regression of cancerous lesions distant from the primary site of radiation delivery and rely upon the induction of immunogenic cell death and consequent systemic anticancer immune activation. Despite the promise of radiation therapy for awaking potent anticancer immune responses, the purposeful harnessing of abscopal effects with radiotherapy remain clinically elusive. In part, failure to fully leverage and clinically implement the promise of radiation-induced abscopal effects stems from limitations associated with existing conventional tumor models which inadequately recapitulate the complexity of malignant transformation and the dynamic nature of tumor immune surveillance. To supplement this existing gap in modeling systems, pet dogs diagnosed with solid tumors including melanoma and osteosarcoma, which are both metastatic and immunogenic in nature, could potentially serve as unique resources for exploring the fundamental underpinnings required for maximizing radiation-induced abscopal effects. Given the spontaneous course of cancer development in the context of operative immune mechanisms, pet dogs treated with radiotherapy for metastatic solid tumors might be leveraged as valuable model systems for realizing the science and best clinical practices necessary to generate potent abscopal effects with anti-metastatic immune activities.

Keywords: immunooncology, comparative oncology, abscopal, immunogenic cell death, radiation, canine, metastases

## SIGNIFICANCE OF A DOG MODEL—STRENGTHS AND LIMITATIONS

### Strengths

Domesticated dogs are second only to human beings in terms of being afflicted with naturally-occurring and inherited diseases, and the purposeful breeding of companion dogs for specific characteristics has produced lineage-specific homogeneity that mimics human demographics such as race or geographic phenotypes (1–4). Dogs acquire genetic diseases as do humans, and consequently might serve as suitable comparative models for conserved pathologies, including certain types of cancer (5, 6). Given that pet dogs often share the same environment and are exposed to similar carcinogens as people, the natural development and evolution of canine tumors can closely parallel those that afflict human beings and share comparable recurrence and metastases patterns. The compressed lifespans of dogs in comparison with humans, combined with the substantial veterinary healthcare dollars spent on pet dogs exceeding \$15 billion annually (7), provide researchers with a robust population of pet dogs available to participate in studies of cancer pathogenesis and the preclinical assessment of investigational therapeutics and medical devices (8–11). Collectively, the shared genetics of specific canine cancers with their human counterparts (12–17), and the high societal value placed upon dogs as companion animals, uniquely and ethically allow pet dogs to serve as potential valuable large animal models for translational cancer research. Particularly, in the era of immunooncology, pet dogs might uniquely serve as ideal parallel tumor models, given the development of spontaneous cancers under competent immune surveillance mechanisms which invariably contributes to shaping of cancer cell immunogenicity and the associated immune topography of the tumor microenvironment (18, 19).

### Limitations

While the recognition of comparative oncologic pathology has been existent for over 50 years (20), the establishment of comparative oncology as a health science discipline by the National Cancer Institute's Center for Cancer Research remains relatively nascent, being formalized in 2003. As such, the purposeful inclusion of pet dogs as parallel cancer models for investigational anticancer immunotherapeutic strategies has only recently begun to bear scientific results in support of the potential model value (21), and has not been maximally leveraged by the scientific cancer research committee given the existence of perceived and true barriers (9), which include heterogeneity of study populations and tumor biology, necessity to conduct adequately powered and prospective clinical trials, and limited availability of diagnostic and therapeutic tools for in-depth scientific investigations. For the study of anticancer immune responses, the diversity and number of commercially available and validated reagents for characterizing immune activation in the domestic canine remain limited in comparison to the existent murine and human reagent toolboxes (22, 23). Additionally, the nuances of immune composition and activation responses in canines is less well-annotated compared to traditional

To expedite the translation of novel immune-based strategies to people with metastatic tumor histologies, the evaluation of experimental therapies in the most highly relevant tumor models should be considered. Besides people, domesticated dogs are also large mammals that develop solid tumors spontaneously that are not only metastatic, but also immunogenic and include canine oral malignant melanoma (OMM) and appendicular osteosarcoma (OS) (29, 30). Importantly, studies demonstrate that these 2 specific solid tumors share similar genetic and histologic features as those found in humans (31–35); suggesting that pet dogs might serve as excellent predictive models for guiding the rational development of immune-based strategies in people with comparable tumor histologies (36).

## IONIZING RADIATION THERAPY

### Radiation Principles and Mechanisms of Cell Death

The biologic responses of cells exposed to radiation traditionally have been categorized into the 5 R's, being Repopulation, Reassortment, Reoxygenation, Repair, and Radiosensitivity. Understandings of these foundational cellular reactions to ionizing radiation have been leveraged to maximize the anticancer activities of radiation therapy (37, 38). The primary target for radiation cellular damage is DNA, and with low linear energy transfer radiation, such as photons and electrons, single strand DNA breaks are created, accumulate, and mimic damage similar to double strand breaks that become difficult, if not impossible, to repair. Consequently, irreparably damaged cells can no longer replicate limitlessly, and the primary cause of cellular death is mitotic catastrophe (39, 40). Irradiated cells can also undergo apoptosis rapidly following radiation exposure with this form of death most relevant to lymphoid cells (39). Other death pathways also play roles in response to radiation, including autophagy and necrosis. Autophagy involves internal degradation of organelles for the promotion of cellular survival and occurs after radiation as a survival mechanism; but can also progress to cellular death and influence inherent radiosensitivity (41, 42). Lastly, by extensive cellular stress through DNA damage, radiation can induce cellular senescence with consequent tumor cell growth arrest (43, 44).

### Radiation-Induced Immunogenic Cell Death and Abscopal Effects

While anticancer activities from radiation have traditionally been ascribed to direct DNA damage to tumor cells, in the era of immunooncology, there has been focused interest to understand the indirect or "out-of-field" immunomodulatory activities induced by radiation therapy. Specifically, a unique form of radiation-induced cell killing called immunogenic cell death (ICD) holds promise for activating systemic immunity against tumor masses distant from the field of radiation delivery (45), a phenomena termed abscopal effect (46). The regressive activity of local irradiation on distant metastatic cells, constituting the abscopal effect, is attributed to an immune-mediated response (47). Given the recognized potential to amplify systemic anticancer immunogenicity following localized radiation, excitement has been garnered by the scientific community to understand and harness the promise of radioimmunotherapy (48, 49).

Mechanistically, ICD has been a focus of radiobiology research and requires activation of the innate immune system through the release of damage-associated molecular patterns (DAMPs) or alarmins, which are released from injured, stressed, or dying cells within the radiation field (50). Scores of different endogenous alarmins derived from cellular organelles and extracellular matrix proteins have been described (51); however, three specific molecules appear to be required for optimal dendritic cell activation and immune priming against malignant cells, specifically being membrane localization of calreticulin and the release of high mobility box group 1 (HMBG1) and adenosine triphosphate into the tumor microenvironment (52). Collectively the expression and secretion of alarmins by dying cells create a localized milieu which exert either "eat me" or "come find me" signals, and are capable of activating innate immune cells exhibiting cognate DAMPs receptors (TLR, RAGE, P2X7), which leads to the priming of cytotoxic T lymphocytes for an adaptive anticancer immune response (53). Given their immune activating properties, the purposeful induction of alarmins within the tumor microenvironment as an in-situ vaccine strategy is actively being investigated (54, 55).

While the elicitation of ICD within the primary tumor microenvironment through ionizing radiation has potential to prime the innate immune system, there remains the necessity for generating sufficient out-of-target tumor responses known as the abscopal effect, especially at sites of metastatic burden that might be unamendable to conventional localized treatment strategies. Despite the documentation of abscopal activities induced by localized radiation therapy in combination with adjunctive treatments (cytokines and chemotherapy), the fraction of human cancer patients that reliably demonstrate abscopal activities sufficient to induce macroscopic tumor regression remains <30% (56). The contextual scenarios (tumor type, host environment, therapeutic combinatorial sequencing) by which abscopal effects can be generated by radiation therapy remain incompletely defined (57, 58). As such, prospective investigations with high-value animal models could accelerate the identification of ideal circumstances to augment the proportion of human cancer patients whom might benefit from the life-extending activities of radiation-induced ICD and associated abscopal effects.

### Opportunity to Optimize Radiation-Induced ICD Protocols

While several recent investigations have discussed the optimal dose and timing of radiation therapy relative to immunologic intervention, no single protocol is clearly superior to others, and the impact of dose rate is relatively unexplored. Given the non-uniformity of various therapeutic radiation regimens for the management of diverse solid tumor histologies, a significant research barrier exists for the thorough characterization of contributory radiation variables required for optimal radiationinduced ICD. While recent meta-analysis has been conducted to "standardize" immune activating potential of radiation treatment protocols through the comparison of biologic effective dose in preclinical models (59), there remains a scientific need for additional prospectively-designed studies inclusive of model systems that more faithfully recapitulate the natural progression of cancer development under immune evolutionary pressures. This "gap" in knowledge given the absence of an ideal experimental model system, is underscored by the rarity of achieving radiation-induced abscopal effects in human cancer patients (56, 60–62). As such, the consistent and reproducible generation of clinically meaningful abscopal effects in most cancer patients remains infrequent and suggests that the current state of understanding regarding radiation-induced immune activation remains incomplete and necessitates the inclusion of complementary innovative modeling systems.

One mechanism to generate new knowledge regarding the feasibility and limitations of radiation-induced ICD and associated abscopal effects could include the rational inclusion of pet dogs with solid tumors. Therapeutic management of cancer in pet dogs parallel the same modalities in human cancer patients, with the inclusion of radiation therapy for controlling localized tumor progression and associated morbidity. Importantly, the repertoire of cognate receptors including toll-like receptors responsible for detecting the presence of pathogens (pathogen associate molecular patterns) and danger signals (damage associated molecular patterns) have been recently characterized in the domestic canine (26, 63–65). With existing tools and knowledge of radiobiology and immunology in the canine species, an opportunity exists to prospectively and systemically evaluate novel radiation-induced ICD strategies in pet dogs that could be translated into life-extending abscopal activities in human cancer patients.

### RELEVANT SOLID TUMORS IN PET DOGS FOR OPTIMIZING RADIATION ABSCOPAL EFFECTS

### Canine Oral Malignant Melanoma (OMM)

Malignant melanoma is a metastatic solid tumor affecting both dogs and people (66), however, the anatomic locations of primary tumors differ, with oral cavity and skin being the primary sites for malignant melanoma in dogs and humans, respectively. In canines, melanoma is considered the most common oral malignancy, accounting for ∼40% of all oral cancers (67). Despite differences in primary anatomic site, prominent molecular drivers of malignancy are conserved between dogs and people, including AKT and MAPK signaling pathways (31).

Effective management of canine OMM requires local treatment strategies, as well as systemic intervention to delay the onset and progression of regional and/or distant metastases

TABLE 1 | Summary of canine melanoma immunogenic strategies.

(68–72). While surgical resection is feasible for some dogs with rostrally-confined primary tumors, most canines are diagnosed with invasive inoperable tumors, and hypofractionated ionizing radiation is instituted for local tumor control (73–75). Radiation therapy, alone or as an adjuvant to marginal resection, can achieve satisfactory local primary tumor control (**Figures 1A,B**), however a substantive fraction of dogs will develop metastatic progression within 6–9 months of diagnosis (67, 68, 76). While the most common site for OMM metastases are regional lymph nodes (77, 78), progression of distant metastases within the pulmonary parenchyma can become life-limiting in dogs that have achieved durable local disease control (67) (**Figures 1C,D**), and the institution of adjuvant cytotoxic agents does not definitively yield any survival benefit (74, 79). As such, no standard-of-care adjuvant therapy in dogs with metastatic OMM exists and creates a unique and ethical opportunity to model novel immunotherapeutic strategies that might not be otherwise possible in human patients. Importantly, commercial reagents for the assessment of immunobiologic endpoints including tumoral expression of PD-L1, tumor-infiltrating lymphocytes, and regulatory T cells have recently been validated in canine tissues (**Figures 1E–H**).

### Clinical Evidence for Canine OMM Immunogenicity

With conservation of certain tumor-associated antigens in both humans and dogs (80–82), canine OMM has been explored as a relevant tumor model in evaluating various immunotherapeutic strategies, in particular tumor vaccine (30). Both autologous and xenogeneic (tyrosinase) vaccines exert measurable anticancer activities in subsets of dogs treated, with objective responses being documented in patients with unsatisfactorily controlled primary tumors, as well as regression of regional and distant metastatic lesions (83–85). In addition to tyrosinase as a therapeutic target, a limited number of investigations have characterized the immunogenic targeting of xenogeneic GP100 and adenoviral CD40L transfection through vaccination strategies; demonstrating immunobiologic activities and clinical benefit in dogs with OMM (**Table 1**) (86, 87).

In addition to vaccines, checkpoint blockade strategies have been recently described in dogs with OMM. Initial studies identified the upregulation of PD-L1 following INF-γ exposure in immortalized canine melanoma cell lines, as well as, PD-L1 expression in 100% (8/8) of spontaneous canine OMM samples (88). A follow-up confirmatory study similarly identified 90% (36/40) OMM samples to express PD-L1, and importantly demonstrated that tumor-infiltrating lymphocytes, both CD4+ and CD8+, expressed PD-1 (89). Expressions of PD-L1 by melanoma cells and PD-1 by TILs, support the potential for melanoma cells to induce T-cell exhaustion as an immunoevasive mechanism. To confirm the functional immunosuppressive activities of PD-L1 expressions in canine OMM, an anti-PD-L1 antibody was evaluated in dogs with OMM, with suggestive evidence for survival time prolongation in four dogs with pulmonary metastasis when compared to historical controls (90). Collectively these clinical investigations support the relevancy of canine OMM as a naturally-occurring model system for testing immunotherapeutic combinations inclusive of other immunomodulatory strategies such as radiation-induced ICD and abscopal activities.

### Canine Appendicular Osteosarcoma (OS)

Osteosarcoma (OS) accounts for 85% of all skeletal tumors in the dog with an estimated 10,000 dogs diagnosed each year (33, 91), and is a disease primarily afflicting the appendicular skeleton of large and giant breed dogs (33). Similarly, OS is the most common primary focal skeletal tumor in people, being the third most frequent cause of cancer in adolescents (92). The comparative similarities at genetic, molecular, and clinical levels shared between canine and pediatric OS are robust (12, 13, 33–35, 93–97); evidence that strongly emphasize the potential value for the utilization of canine OS to guide investigations related to pathogenesis and novel therapeutic discovery (98).

The biologic behavior of OS is aggressive, starting within the local bone microenvironment but then involving distant organs because of metastatic progression. Although 15% of dogs and 20% of people present with detectable lung metastases, the development of metastatic foci in the absence of chemotherapy is 90% within 1 year for dogs and 80% within 2 years for people (99, 100). While the institution of chemotherapy for OS patients has tripled the cure rate of people (20 → 65%) and doubled the survival time of dogs (130 → 270 days), no substantive improvement in long-term outcomes has been achieved for either species over the past 2 decades despite the institution of dose intensification strategies (101, 102). Given the current therapeutic ceiling, there is clinical need to explore alternative adjuvant therapies that might improve metastatic disease control.

Because the cure rate for canine OS remains <10% 3-years post diagnosis (103), the palliative management of primary tumor malignant osteolysis and associated pain is considered an acceptable treatment option in veterinary medicine (104). Similar to skeletal metastasis in humans, ionizing radiation alone or with bisphosphonates is considered effective for attenuating pathologic bone resorption and associated pain syndromes in affected dogs (105–111), and provides a durable therapeutic window of acceptable analgesia lasting from 3

to 12 months, whereby it is possible to serially monitor for the development, progression, or regression of distant pulmonary metastases. Prospective assessment of combinatorial strategies inclusive of radiation and other immunostimulatory therapies to amplify tumoral lymphocyte infiltrates such as ICD-inducing anthracyclines, toll-like receptor agonists, and checkpoint blocking antibodies which maximally generate robust abscopal effects could be leveraged to guide translational studies in human patients (**Figure 2**).

### Clinical Evidence for Canine OS Immunogenicity

Scientific and clinical evidence supports OS to be immunogenic in dogs and humans (29, 112), and strategies that amplify anticancer immunity would be expected to improve longterm outcomes. In dogs, investigations have demonstrated immune activation as an effective strategy for either regressing macroscopic metastases or delaying micrometastatic disease progression. For macroscopic disease, inhalation therapy with liposome interleukin-2 demonstrated the capacity to activate immune cells with consequent regression of measurable pulmonary metastases (113, 114). In the setting of microscopic disease, dogs that develop post-operative wound infection after limb-spare surgery experience prolongation to pulmonary metastases development, with survival times being doubled in dogs that develop osteomyelitis (115, 116), and mechanistically localized infectious inflammation has been linked to NK cell and macrophage activation with consequent mediation of systemic anticancer effects (117). Similarly, L-MTP-PE, a synthetic lipophilic glycopeptide capable of activating monocytes and macrophages to a

tumoricidal state, when administered to dogs with OS increases survival time, and underscores the key participation of innate immune cell activation for curbing metastatic progression (118, 119). Lastly, intravenous delivery of a genetically modified Listeria monocytogenes to OS-bearing dogs exerts promising anticancer immune activities and extends survival times (120). Collectively, these clinical investigations support the feasibility of stimulating immune effector cells to regress macroscopic and microscopic metastatic disease burdens in dogs diagnosed with OS.

### Emerging Abscopal Modeling in Canine OMM and OS

While existing aggregate data for validating radiation-induced ICD and abscopal activities in pet dogs with cancer remains limited, experimental data is emerging to support the prospective evaluation of hypofractionated radiation therapy for augmenting immune responses. Recently, combinatorial strategies inclusive of ionizing radiation, hyperthermia, and intratumorally delivered virus-like nanoparticle-based therapies have been evaluated in canine OMM, and demonstrate the capacity to elicit immunogenic changes within the localized tumor microenvironment including the promotion TILs into the primary tumor (121, 122). In another investigation conducted in dogs with OMM, abscopal effects were documented in dogs treated with a combination of localized radiation therapy, intratumoral CpG ODN, and an indolamine-2,3-dioxygenase inhibitor (123). For canine OS, combining radiation and immunotherapy has been recently explored in a first-in-dog trial of autologous natural killer (NK) cells (124). In this study, OSbearing dogs were treated with a coarsely fractionated radiation protocol consisting of 9 Gy once weekly for 4 treatments, with NK cells being harvested and expanded, and then delivered back to dogs by intratumoral injection following the completion of radiation therapy. Of the 10 dogs treated, 5 remained metastasisfree at 6 months, and one had regression of a suspicious pulmonary nodule detected at the time of diagnosis.

### FUTURE DIRECTIONS AND CONCLUSIONS

Dogs diagnosed with naturally-occurring cancers of comparative relevance can serve as biology-rich models of disease. If

### REFERENCES

leveraged appropriately, the inclusion of pet dogs can accelerate the discovery of optimal combinations of radiation and immunotherapies which robustly and consistently elicit lifeextending abscopal effects. With the availability of linear accelerator-based radiation facilities in veterinary centers analogous to human hospitals, coupled with the development of dog-specific immune-based therapies including vaccines, monoclonal antibodies, and CAR-T technologies, the purposeful inclusion of pet dogs with immunogenic tumors should be seriously contemplated as a unique strategy to aid in defining the limits and benefits of radiation-induced abscopal activities.

The scientific development and clinical assessment of novel immunotherapeutic strategies are rapidly growing areas in veterinary medicine and have demonstrated promise in the settings of canine OMM and OS. Given the conserved biology of these two immunogenic solid tumors between dogs and people, unique opportunities exist collectively for human and veterinary researchers to pilot and validate innovative immune strategies inclusive of radiation therapy in efforts to harness the promise of abscopal anticancer activities.

### AUTHOR CONTRIBUTIONS

TF and KS project conception and manuscript authorship.

### FUNDING

This mini-review was supported by Morris Animal Foundation, D19CA-064.

### ACKNOWLEDGMENTS

The authors wish to acknowledge Renee Walker and Drs. Michael Kent (UC Davis) and Jonathan Samuelson (UIUC) for technical assistance and provision of images.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Fan and Selting. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Development of Severe Combined Immunodeficient (SCID) Pig Models for Translational Cancer Modeling: Future Insights on How Humanized SCID Pigs Can Improve Preclinical Cancer Research

Adeline N. Boettcher 1 , Crystal L. Loving2 , Joan E. Cunnick 1 and Christopher K. Tuggle1 \*

*1 Department of Animal Science, Iowa State University, Ames, IA, United States, 2 Food Safety and Enteric Pathogens Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, IA, United States*

#### Edited by:

*Kyle Schachtschneider, University of Illinois at Chicago, United States*

#### Reviewed by:

*Saraswati Sukumar, Johns Hopkins University, United States Qingsheng Li, University of Nebraska-Lincoln, United States*

> \*Correspondence: *Christopher K. Tuggle<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Molecular and Cellular Oncology, a section of the journal Frontiers in Oncology*

Received: *27 August 2018* Accepted: *09 November 2018* Published: *30 November 2018*

#### Citation:

*Boettcher AN, Loving CL, Cunnick JE and Tuggle CK (2018) Development of Severe Combined Immunodeficient (SCID) Pig Models for Translational Cancer Modeling: Future Insights on How Humanized SCID Pigs Can Improve Preclinical Cancer Research. Front. Oncol. 8:559. doi: 10.3389/fonc.2018.00559* Within the last decade there have been several severe combined immunodeficient (SCID) pig models discovered or genetically engineered. The animals have mutations in *ARTEMIS*, *IL2RG*, or *RAG1/2* genes, or combinations thereof, providing SCID pigs with NK cells, but deficient in T and B cells, or deficient in NK, T, and B cells for research studies. Biocontainment facilities and positive pressure isolators are developed to limit pathogen exposure and prolong the life of SCID pigs. Raising SCID pigs in such facilities allows for completion of long-term studies such as xenotransplantation of human cells. Ectopically injected human cancer cell lines develop into tumors in SCID pigs, thus providing a human-sized *in vivo* model for evaluating imaging methods to improve cancer detection and therapeutic research and development. Immunocompromised pigs have the potential to be immunologically humanized by xenotransplantation with human hematopoietic stem cells, peripheral blood leukocytes, or fetal tissue. These cells can be introduced through various routes including injection into fetal liver or the intraperitoneal (IP) space, or into piglets by intravenous, IP, and intraosseous administration. The development and maintenance of transplanted human immune cells would be initially (at least) dependent on immune signaling from swine cells. Compared to mice, swine share higher homology in immune related genes with humans. We hypothesize that the SCID pig may be able to support improved engraftment and differentiation of a wide range of human immune cells as compared to equivalent mouse models. Humanization of SCID pigs would thus provide a valuable model system for researchers to study interactions between human tumor and human immune cells. Additionally, as the SCID pig model is further developed, it may be possible to develop patient-derived xenograft models for individualized therapy and drug testing. We thus theorize that the individualized therapeutic approach would be significantly improved with a humanized SCID pig due to similarities in size, metabolism, and physiology. In all, porcine SCID models have significant potential as an excellent preclinical animal model for therapeutic testing.

Keywords: severe combined immunodeficiency, swine, humanization, cancer, xenograft, pre-clinical, animal model

### INTRODUCTION

A new field of personalized medicine has been evolving over the last decade, especially with respect to advances in individualized cancer therapies, ranging from T cell and NK cell immunotherapies, targeted monoclonal antibody therapy, and newly developed small molecule drugs. As progress is made toward the development of cancer therapies, it is critical that preclinical animal models can dependably represent human responses to drugs. Presently, mice are the most commonly used model for preclinical animal drug trials (1). However, many preclinical cancer drug trials that succeed in mice fail in humans due to vast differences in physiology, metabolic processes, and size (2, 3). The drug development process is intensive; on average, 12 years of research and \$1–2 billion is required to bring a new drug to market (4, 5). To maximize the efficiency of preclinical drug and therapy testing, large animal models that better parallel human physiology are needed.

Mice with severe combined immunodeficiency (SCID) are an extremely versatile animal model for the field of cancer biology, although they pose significant limitations. The ability to engraft SCID mice with a human immune and/or cancer cell lines has made them an invaluable model for research (6, 7). Although mice are important for initial studies in different cancer fields, they are often not good models for specific aspects of human oncology (2, 8). Limitations of mouse models of cancer include small size, difficulties in modeling human tumor heterogeneity (9) and metabolic differences to humans (10, 11).

Large animal models can be more costly than murine studies, thus murine studies remain valuable for first line screens. However, testing in larger animal models is warranted to better predict outcomes in human and should be used in follow-up studies as an alternative animal model (12). Immunocompetent and SCID pigs are now being developed for human disease research purposes (13–18). Swine are more similar to humans with respect to size, anatomy, genetics, and immunology, therefore immunodeficient pigs may be a superior animal model for preclinical testing of cancer therapeutics (19–21).

Within the last decade there have been numerous SCID pig models created (16–18, 22–28) or discovered (29, 30). One of the hurdles to working with SCID pigs is maintaining viability due to susceptibility to disease. The use of positive-pressure biocontainment facilities (31) and standard animal isolators (27) have improved SCID pig health and viability. The ability to house immunodeficient pigs in a controlled environment increases their lifespan allowing them to be utilized for long-term biomedical research. Pigs are comparable in size to humans, have more similar metabolism to humans than mice (32, 33), and can be transplanted with larger human tumors.

In this review we describe the different SCID pig models that have been reported in recent years, as well as published methods established to raise SCID pigs for use in long-term research trials of 6 months or more. We describe the importance of human tumor or cancer cell xenotransplantation and how researchers can utilize immunodeficient pigs for translational studies relevant to human patients. In addition to tumor xenografts, the SCID pig has the potential to be engrafted with a human immune system, or "humanized," just as numerous SCID mouse models have been humanized. While there is no published research on the development of a humanized SCID pig, substantial progress is being made toward this endeavor. We describe the different methods of humanization that could be used in SCID pigs, including fetal liver and intraperitoneal (IP) injections, as well as intravenous (IV), IP, and intraosseous (IO) injection in piglets. Despite the early developmental stage for humanized SCID pigs, the SCID pig has vast potential to be utilized for translational oncology. Our overarching hypothesis in this review is that porcine SCID models will be more translational than mouse models for oncology research in the future.

  **Figure 1** shows the genetic and molecular mechanisms for the mutations described below that cause SCID, and **Figure 2** shows the differentiation step blocked by each of these mutations.

The first SCID pig was described in 2012 (13) after a serendipitous discovery in an infection study (29). To confirm the lack of a functional immune system, these SCID pigs were transplanted with human cancer cell lines. Injected cells were not rejected and developed into tumors in the SCID pigs (13). After further analysis, it was found that the discovered SCID pigs had two naturally occurring mutations in two separate alleles within the Artemis (DCLRE1C) gene, which leads to SCID either in the homozygous or compound heterozygous state (30).

Artemis is required for DNA repair during T and B cell development. Specifically, during the process of VDJ recombination, after RAG1/2 nucleases cleave DNA at the RSS sequences flanking V, J (and sometimes D) segments (34), a hairpin loop then forms at the end of the double stranded break (DSB). Ku70/80 proteins are recruited to the area of the DSB along with Artemis protein, which is responsible for cleaving the hairpin loop so it can be ligated by Ligase IV (35). Without functional Artemis, these hairpins are not cleaved, and functional V, D, and J joins cannot be made. Lack of Artemis function leads to a cellular profile in which T and B cells are deficient, but NK cells develop (T− B − NK+) and are functional (29, 30, 36). Homozygous or compound heterozygous Artemis pigs can be

**Abbreviations:** SCID, severe combined immunodeficiency; IL2Rγ, Interleukin 2 receptor gamma; RAG, recombination activating gene; Hematopoietic stem cells, HSCs; PBL, peripheral blood leukocytes; VDJ, variable, diversity, and joining; TCR, T cell receptor; BCR, B cell receptor; DSB, double stranded break; NSG, NOD-SCID-IL2Rγ; IP, intraperitoneal; IV, intravenous; TREC, T cell receptor excision circles; GVHD; graft vs. host disease; BLT, bone marrow, liver, and thymus; CAR, chimeric antigen receptor; CRS, cytokine release syndrome; PET, positron emission tomography; MRI, magnetic resonance imaging; CT, computer tomography; US, ultrasound; PDX, patient derived xenograft; CDX, cell derived xenograft.

raised to 6 months of age in biocontainment facilities developed at Iowa State University [31, unpublished observation].

Another SCID pig was also described in 2012 with an engineered mutation within the IL2RG gene (16). In humans and mice, the IL2 receptor γ (IL2Rγ) subunit is required for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 signaling (37). The IL2RG gene is on the X chromosome in mammals and the receptor is expressed on lymphoid cells, including developing cells. The cytokines noted are required for proper lymphoid development, and thus deletion of the IL2Rγ subunit disrupts development of T and NK cells, and B cells to a variable extent (38, 39). The cellular phenotype of these IL2RG knockout pigs was T− B + NK−, similar to humans (38, 39). B cells in IL2RG knockout SCID pigs were not able to secrete immunoglobulin nor class switch due to absence of helper T cells (16). Interestingly, cloned heterozygous IL2RG+/− females exhibited SCID-like phenotypes, which was attributed to aberrant X-inactivation. These females were raised to sexual maturity and crossed with WT males; female IL2RG+/− offspring from this cross phenotypically resembled WT animals (16). This finding emphasizes the importance of monitoring for SCID phenotype status in cloned piglets even if the expected outcome is a carrier animal. Other groups have also introduced mutations in the IL2RG gene by CRISPR/Cas9 (17) and zinc finger nuclease (18) methods, and the resulting pigs also displayed cellular phenotypes of T−B +NK−. Animals in these studies were raised in conventional settings and had lifespans that ranged from 12 days to 7 weeks (16–18).

The recombination activating genes, RAG1 and RAG2, have previously been mutated to create pig models. They code for subunits of a nuclease (RAG1/2), that is involved in VDJ recombination required for T and B cell receptor (TCR and BCR, respectively) generation (40). Without functional RAG1/2 nuclease, VDJ recombination does not initiate, and T and B cells do not develop (41, 42). Homozygous or biallelic RAG1 or RAG2 SCID pigs lacked IgM+ B cells and CD3+ cells in peripheral blood (22, 23, 25). NK cells were present in these animals and were classified as either CD3− CD8α + (22) or CD16+ CD8α + (25). RAG knockout pigs were generated with either TALENs (22, 24), gene targeting vectors (25), or CRISPR/Cas9 (26) mutagenesis methods. Previous RAG1 or RAG2 mutant SCID pigs have been raised to 29 days (22, 24) to 12 weeks (25) of age in conventional housing.

Once single mutant pigs were established, research groups began to introduce mutations in both VDJ recombination pathway genes (RAG1/2 or ARTEMIS) and IL2RG to produce pigs that lacked innate and adaptive immune function, generating T − B − NK−/lo SCID pigs (26, 28). Double-mutant pigs are an important animal model to develop, as rodent models of SCID mice lacking NK cells, as well as T and B cells, engraft human cells better than T−B − NK+ models (43). It is therefore of interest to generate a T− B − NK− SCID pig model for humanization studies. In 2016, RAG2/IL2RG knock out piglets were generated and used in pathogenesis study with human norovirus (26). RAG2/IL2RG SCID pigs lacked T and B cells, and there were decreased numbers of NK cells compared to controls. The presence of some NK cells was attributed to a hypomorphic mutation within IL2Rγ (26). Our group has recently engineered a complete IL2RG knockout that was introduced into an ARTEMIS null genetic background resulting in SCID pigs that lack T, B, and NK cells (28).

### Methods for SCID Pig Rearing

One of the difficulties to overcome when using SCID pigs in research is maintaining animal viability. SCID pigs raised in conventional settings typically succumb to disease between 6 and 12 weeks of age [unpublished observation, 17]. Biocontainment facilities have been specifically designed to limit exposure of Iowa State University's ARTEMIS−/− SCID pigs to any micro-organisms (**Figure 3A**). These rooms have positive-pressure HEPA filtered air flow into a containment bubble and all water entering the bubble is UV irradiated and filtered through a 0.5µm filter. Personnel entering the bubble wear appropriate garments to limit introduction of organisms into the room, including room dedicated protective suits, hair net, surgical mask, gloves and rubber boots (31). Piglets are derived either by snatch farrowing (caught in a sterile towel as they are delivered vaginally) or by cesarean section and are transferred immediately into

a sterilized bubble. Piglets are immediately fed pasteurized colostrum for the transfer of maternal immunoglobulin (44), fed sterile milk replacer for 21 days, and then transitioned to irradiated feed, which is continued throughout life (31). Specific pathogen-free (SPF) ARTEMIS+/− carrier females have been raised to sexual maturity and are able to naturally farrow ARTEMIS−/− SCID litters within the ISU bubble facilities (**Figure 3B**). ARTEMIS mutant SCID pigs can be successfully reared to 6 months of age in these facilities (unpublished observation).

Survivability of previous IL2RG knock out pigs has varied from 2 to 7 weeks (16) and derivation of animals and available housing likely impacts outcome. Recently Hara et al. (27) used small isolators and developed piglet delivery protocols to help extend the lifespan of IL2RG knock out SCID pigs. To achieve this goal, excised uteruses were brought into isolators units, piglets were delivered, and reared within these isolators. One SCID piglet raised in the isolators was raised to a planned endpoint of 12 weeks of age without incidence of bacterial or fungal disease (27).

FIGURE 2 | Lymphoid development and relevant SCID pig mutations. Mutations in Artemis, RAG1/2, and IL2Rγ leads to SCID in pigs. Artemis and Rag1/2 are active in Pro-B and -T cells during differentiation. IL2Rγ is required at an earlier stage of development than RAG1/2 and Artemis. NK cells and T cells both require cytokine signaling through IL2Rγ early in differentiation. Mutations in IL2Rγ prevent differentiation of T and B cells. Mouse B cells appear to rely on IL2Rγ signaling more than human and pig B cells. B cells can still develop in humans and pigs with mutations in IL2Rγ, although they are mostly non-functional due to the absence of helper T cells.

### SCID PIG CANCER XENOTRANSPLANTATION STUDIES

### Existing Immortal Cell Lines Develop Into Tumors in SCID Pigs

Since the generation of SCID pigs is so recent, there are only a few studies that have been published on the ability of SCID pigs to accept human xenografts. The first SCID pig xenograft study involved the transplantation of human melanoma (A375SM) and pancreatic carcinoma cell (PANC-1) into the ear tissue of ARTEMIS−/− SCID pigs (13). All SCID pigs receiving cancer cells developed tumors at the site of injection, thus establishing an orthotopic model of melanoma that could be studied further (13). Additionally, the ability of ovarian carcinoma cell line OSPC-ARK1 to develop tumors in ARTEMIS−/− SCID pigs was explored. SCID pigs were injected in the ear and neck muscles with OSPC-ARK1 cells and subsequently monitored for tumor development. In 3 of the 4 SCID pigs injected, tumors developed within 30 days, with a shortest time of 7 days to palpable tumors. Biopsy samples revealed the ovarian tumors in SCID pigs expressed diagnostic markers commonly used in human cancer diagnoses, and tumors in SCID pigs resembled human tumors (45).

Pigs biallelic for RAG2 mutations can engraft human induced pluripotent stem cells (iPSCs). Injected iPSCs developed teratomas that represented endoderm, mesoderm and ectoderm tissues (24). Teratomas were grossly visible 12 days after cell inoculation for one recipient; and about 7.5 weeks in the other recipient. Histological analysis revealed CD34+ and CD45+ cells developed in the teratoma, (24), indicating that human immune lineage can survive and differentiate in RAG2 knockout pigs. This important finding indicates that SCID pigs can accept various types of human xenografts. In a follow-up study, PERFORIN, and RAG2 double knock out (Pfp/RAG2 dKO) mice and RAG2 knock out pigs were compared for their ability to engraft human iPSCs. The RAG2−/− pigs developed teratomas from injected iPSCs at a higher rate than the Pfp/RAG2 dKO mice. Human teratomas that developed in the RAG2 knockout SCID pigs also had a higher prevalence of CD45+ and CD34+ cells in the teratoma than in SCID mice (46). Thus, the in vivo environment in pigs supports the growth and differentiation of human cells, and in some instances, is an improved system over SCID mice.

### PORCINE IMMUNOLOGICAL SIMILARITIES TO HUMAN

Several aspects of the pig immune system are more similar to humans than mice, providing another advantage of swine models for research (39). Humans and pigs have higher sequence orthology for immune-related genes (termed the "immunome") than humans and mice (20). Immunome-specific gene family expansions, a measure of evolutionary divergence, have occurred in pig relative to human at half the rate detected in mouse or cow (20), and pigs have significantly fewer unique genes not found in humans when compared to unique gene abundance in cow or mouse (**Figure 4**). Additional analyses have further expanded human and pig similarities, although absence of two inflammasome gene families have also been found uniquely in the pig genome (47). As well as immunome structural similarities, immune responses are highly comparable between human and pig [reviewed in (41)]. For example, the transcriptomic response to lipopolysaccharide of pig macrophages in vitro is more similar to human responses as compared to mice. Specifically, clusters of genes with IDO1 as hub were detected in human and pig macrophage responses, but not in mice, while a NOS2A-related gene cluster was only found in the mouse macrophage LPS response (48).

Human hematopoietic stem cell (HSC) development in swine for humanizing pigs will be dependent on swine cytokine signaling. Hence, it is important to determine the cross reactivity of porcine cytokines with human cells. Protein sequence analysis shows that swine share more homology in cytokines involved in hematopoiesis with humans than mice (**Figure 5**; **Supplemental Table 1**), which suggests that certain human

lineages may differentiate with greater success in SCID pigs than in SCID mouse models.

### ROUTES FOR HUMANIZATION AND APPLICATIONS

Given the high similarity of swine and human immune genes, we would anticipate that human HSCs transferred into SCID pigs would successfully engraft and differentiate into representative human immune cell types. Current building of swine SCID models relies heavily on translating methods used for mouse humanization to generate new humanized SCID pig models. To humanize the mouse, three different approaches are utilized (6, 7). These methods include transfer of purified human CD34+ stem cells, peripheral blood leukocytes (PBLs), or transfer of fetal bone marrow, liver, spleen, and lymph node tissues. Just as in SCID mouse models, these same approaches and cell types can be investigated as methods to humanize SCID pigs. The pig immune signaling molecules that support engraftment are expected to be similar to humans, thus we expect successful development of human immune cells.

Currently the NOD-SCID-IL2Rγ (NSG) knockout mouse is the gold standard model for humanization. The Sirpa allele in the NOD background contains polymorphisms that allow the encoded Sirpa protein to bind to human CD47, which then sends a inhibitory signal that prevents phagocytosis of human cells (49, 50). Swine SIRPA also binds to human CD47 (51), so we speculate that porcine SIRPA-dependent phagocytosis of human cells would not be a barrier to SCID pig humanization.

The following sections describe previous humanization methods performed in SCID mice and other large animal models, and how these methods can be utilized to humanize SCID pigs. **Figure 6** shows an overview of different human immune cell

types and anatomical injection sites for SCID pig humanization. Past studies utilizing injection of human HSCs or human induced pluripotent stem cells into large animal models are presented in **Table 2.**

### CD34+ Cell Injection via Fetal Liver and Intraperitoneal Space

Successful humanization of SCID pigs will require that human HSC be injected into sites of hematopoiesis in the pig. During gestation the initial location of hematopoiesis is the yolk sac (58). As gestation continues, the fetal liver becomes the site of hematopoiesis, typically around the beginning of the second trimester (59–63). During swine gestation, hematopoiesis begins at day 30 in the fetal liver (62). Intrauterine injection of human hematopoietic cells during the fetal liver phase of hematopoiesis would provide a rich environment for human stem cells to engraft and differentiate (64), as supporting cells in the fetal liver niche express c-Kit, CD34, CXCL12, and NOTCH (59). Additionally cell subsets in the fetal liver can promote hematopoiesis, such as CD34lo CD133lo cells that have been described in human (65). Differentiated human cells that develop in the SCID pig liver may also migrate to the bone marrow around the same time as other developing swine immune cells, which may increase the ability of human immune progenitors to engraft within the SCID pig bone marrow. Fewer human cells would be required for the fetal liver injection strategy when compared to the number of cells required to engraft a fully developed piglet. Taken together, we hypothesize that fetal injection of human hematopoietic stem cells will likely lead to the highest levels of engraftment compared to other methods described in later sections.

The first study involving in utero injection of human cells into a large animal was performed by Zanjani et al. (52). Human fetal liver cells were injected into the IP space of fetal sheep at days 48–54 gestation (145 day term) through the uterine wall. The recipient sheep were immunocompetent, but pre-immune at this stage of development. Two of the derived sheep were raised to 15 months of age, and human CD3+, CD16+, and CD20+ cells were still in circulation, albeit at very low frequencies (52). Other studies involving the transplantation of human CD34+ cells in the fetal liver of pre-immune sheep have resulted in similarly low levels of human cell engraftment and differentiation (55, 57).

In addition to sheep, in utero injection of human CD34+ cells have been performed in pre-immunocompetent conventional pigs. The first was described in 2003 (53) with the injection of cord blood derived CD34+ cells into the IP space of preimmune fetal piglets at ∼40 days of gestation (114 day term). Populations of human CD3+ cells were detected in the thymus, CD19+ cells and myeloid cells also developed de novo in the pig, in as short as 40 days post-injection. Additionally, human CD34+ CD45+ cells were isolated from pig bone marrow 120 days after transplantation and were subsequently transplanted into SCID mice with successful engraftment of human cells observed. This result indicates that the pig bone marrow environment is able to support the development of functional human HSCs (53).

Humanization of pigs could serve as a source of human T cells for immunotherapeutic use. Ogle et al. (56) depleted CD3+ cells from human bone marrow or cord blood and injected into the IP space of fetal piglets at 40–43 days of gestation. Human T, B, macrophages, and NK cells were detected in peripheral blood of piglets using RT-PCR by amplification of CD3, CD19, CD14, and CD16/CD56, respectively. In order to determine if the human T cells had developed de novo, blood was analyzed for the presence of human T cell receptor excision circles (TREC). Human TRECs were observed at a level that suggested new human T cells had developed in the swine thymus (56). Similar,

FIGURE 6 | Cell types and routes of injection for SCID pig immunological humanization. Swine can be injected with human cells at two different developmental stages. During gestation, fetal piglets at ∼40 days of gestation can be injected with human CD34+ stem cells within either the liver or intraperitoneal space via ultrasound guidance. Newborn piglets can also be injected with human CD34+ stem cells through either intravenous or intraosseous routes. PBLs can also be injected via intravenous injection. Fetal tissues including bone marrow, liver, thymus, or spleen can be transplanted within the abdomen, potentially under the kidney capsule as is done with SCID mice.

TABLE 2 | Previously described human stem cell injection studies in swine and sheep.

*PI, Pre-immunocompetent; ESC, embyronic stem cells; iPSC, induced pluripotent stem cells.*

studies were performed in which fetal swine were injected with human T cell depleted bone marrow (54) or T cell depleted cord blood (66), in which human cell engraftment was observed. In all, these studies show that human T cells can develop de novo when human HSC are injected into fetal swine.

Successful engraftment of SCID pigs utilizing in utero injections requires consideration of timing and surgical procedures. We hypothesize that a humanized SCID pig could be developed via in utero injection of human CD34+ cells within the fetal liver or IP space at ∼40 days of gestation. We have described detailed laparotomy protocols that can be followed for procedures involving stem cell injection into fetal IP space and livers [(67); **Figure 7**]. The level of human cell hematopoiesis in a SCID pig model has yet to be determined, however it is expected that engraftment would be comparable to that described for immunocompetent animals. Given the lack of pig immune cell development in pigs with SCID, the available niches for human progenitor cells to develop in the bone marrow and thymus would be increased.

### Peripheral Blood Leukocyte Injection via Intravenous or Intraperitoneal Routes

In 1988, the first humanized mouse models were generated in efforts to investigate the AIDS virus interaction with its human host. One of these models described the injection of human PBLs into the IP space of SCID mice (68). Mice were injected by the IP or IV routes with 10–90 million human PBLs (termed hu-PBL SCID mice). IV injection was deemed ineffective in mice, likely due to the difficulty of proper IV administration in a mouse. Human cells injected IP in mice were able to migrate to the spleen, lymph nodes, and were also detected in peripheral blood; 4 weeks post IP injection very few human PBL were detected in the peritoneal space. Mice were vaccinated with tetanus toxoid, which PBL donors were known to be immune. Eight of 10 animals injected with PBLs produced human immunoglobulin against tetanus toxoid which supported that human helper T cells and B cells were functional in the hu-PBL SCID mice. Human CD14+ monocytes were also present in the spleens of mice 8 weeks post transplantation (68). These hu-PBL SCID

mice are utilized in a variety of different fields including HIV (69–71), cancer (72, 73), basic immunology (74, 75), and atopic dermatitis (76).

Hu-PBL-SCID pigs could be generated by IV or IP injection of human PBLs into SCID pigs. IV injection of human cells have been deemed ineffective for engraftment in mice. However, tail veins are typically used in mice, which are difficult to properly inject. Piglets have large and visible ear, cephalic, and saphenous veins that are easily accessible. A limitation of human PBL injections in pigs could be the amount of cells required relative to the number of cells injected into mice. Mice are typically 20 g (0.02 kg), while a typical newborn piglet weighs about 1–2 kg. In previous studies, the minimum amount of human PBLs injected into mice is about 10 million cells, which scales up to 0.5–1 billion in a piglet. However, there are strategies to overcome the cell number limitation. One source for human PBLs could be leukoreduction system chambers (LRSCs), which are utilized by blood banks to remove PBLs during plateletpheresis. During a normal collection of platelets from a donor, ∼2 billion PBLs can be obtained from LRSCs (77). Another approach is matching a human with a SCID pig and performing repeat PBL injections from the same human donor. Also, it is possible that the number of human PBLs required for successful engraftment of SCID pigs would not be as high as calculated from murine studies. Given that methods to obtain large numbers of PBLs are available, the number limitation is not expected to prevent development of a hu-PBL-SCID pig.

One consideration for using a SCID pig injected with human PBLs is that these animals will eventually develop graft vs. host disease (GVHD). SCID mice injected with human PBLs develop GVHD ∼3–11 weeks after injection (78) while it takes 14–16 weeks in SCID rats (79). It is currently unknown how long it would take SCID pigs to develop GVHD after human PBL cell transplantation, as well as how the cellular dose would impact the GVHD time frame. This is a question that will need to be addressed as this model is developed. Another important question that will need to be addressed in developing this model is the time period required for human PBL engraftment within the SCID pig.

One benefit of the PBL model is that it could be used for short term studies in SCID pigs. SCID pigs raised in conventional settings can typically survive to 6 weeks of age. If piglets are injected with human PBLs shortly after birth (1– 5 days), this would give researchers ∼a 6 week window to perform experiments. It may also be appropriate to administer immunosuppressive drugs during this period of time to reduce the effects of GVHD.

### CD34+ Cell Injection via Intraosseous or Intravenous Routes

Another route for humanization is through the injection of purified human CD34+ HSCs into live-born piglets. We have previously performed bone marrow transplantations (BMT) on our SCID pigs through IV injection of unfractionated pig bone marrow cells (80). One hypothesis is that human HSC could be administered in the same way to generate a humanized SCID pig. Typically in pig to pig bone marrow transplants, it takes ∼10 weeks to observe a moderate increase in the number of circulating porcine lymphocytes (80). We hypothesize that human engraftment and de novo development of human cells would require at least 10 weeks to observe human cells in circulation based on pig to pig BMT observations. It may be of value to compare cell dosages and engraftment rates of human and pig HSC in SCID pigs. IV injection of human HSC is much less invasive than fetal injections, however it may take longer to achieve engraftment and differentiation of human cells.

Another method of human HSC administration is through intraosseous (IO) injection. IO injection of stem cells and mesenchymal stem cells have previously been performed in SCID mice (81), dogs (82, 83), and pigs (84). IO injection is also a method for bone marrow transplantation in humans (85). It is hypothesized that IO injections are preferable over IV injections due to stem cell trapping in pulmonary tissue, which is often observed in IV injections (86, 87). In addition, IO administration introduces cells to the site within which they would differentiate. Protocols have also been developed for the delivery of various substances though IO injection in swine (84, 88, 89). IO injection of human CD34+ cells into SCID pigs is therefore another potential route for studying engraftment and humanization models.

### Implantation of Human Fetal Bone Marrow, Thymus, and Liver Tissues

Another potential method for humanization of SCID pigs is through the transplantation of human fetal liver, thymus, lymph node, and spleen tissue, as has been previously performed in mice (90). Such human lymphoid tissues can be transplanted into mice either by implantation under the kidney capsule or IV injection of a cellular suspension. Mice transplanted with human lymphoid tissues appear to have immunological protection, as the lifespan of transplanted mice can be extended to 17 months, compared to 4 months for non-transplanted mice. Mice injected with both human thymic and fetal liver cells developed human T and IgG secreting B cells (90). The chimeric mice with human bone marrow, liver and thymus (BLT) are used to study interactions between human immune cells and patient derived melanomas (91).

De novo development of human T cells within the pig requires that human T cells can differentiate within the swine thymus. Transplantation studies show that the porcine thymus supports human T cell development, as mature human T cells develop in athymic mice transplanted with porcine thymus and human HSCs (92, 93). Human T cell development within the swine thymus is particularly important for long term studies because this would allow newly differentiated human T cells to develop tolerance to pig antigens. Human thymic tissue could also be transplanted into SCID pigs for human HLA restricted T cell development. Development of GVHD is observed in mice humanized with fetal bone marrow, liver, and thymic tissue (94), potentially due to human thymus dependent T cell development. Depending on the experimental question being addressed, transplantation of a human thymus may be a preferred method in humanizing SCID pigs.

One issue with generating BLT humanization models is the limited fetal tissue availability, as well as ethical implications. Smith et al. described a way to circumvent these issues by propagating and expanding BLT tissues in one mouse and then transplanting into 4–5 other mice (95). SCID pigs could be useful in this regard as human tissues would have the potential to grow to a large enough size that they could be transplanted again into a second set of animals.

### FUTURE OUTLOOK ON THE UTILIZATION OF SCID PIGS FOR CANCER THERAPIES AND RESEARCH

### Humanized SCID Pigs for CAR-T and CAR-NK Cell Therapy Research

Chimeric antigen receptor (CAR) T and NK cells have been developed in recent years as a cancer immunotherapy. CAR-T cells targeted against CD19 for patients with B cell lymphomas and leukemias (96) have been approved by the FDA for therapeutic use (97, 98). One of the issues associated with CAR-T cells is that they can persist and be activated for long periods of time in the body, causing cytokine release syndrome (CRS). Symptoms of CRS manifest as fatigue, fever, nausea, cardiac failure, among other symptoms (99). CAR-NK therapies are being developed to overcome some of the issues associated with CAR-T cell therapy. Protocols have been developed to isolate NK cells from cord blood and expanded for use in patients. NK cells do not persist for long periods of time in vivo after infusion (100), do not cause GVHD, and can recognize tumor targets through intrinsic receptors (101). If SCID pigs can successfully develop human NK cells de novo, humanized SCID pig blood could be a source of NK cells. Six month old ARTEMIS−/− Yorkshire SCID pigs are ∼85 kg (personal observation), and thus according to IACUC guidelines, up to 1.2 L of blood could be collected for human NK cell isolation and used for CAR therapy research.

We envision several applications for a hu-PBL SCID pig in testing cell-based immunotherapies. As more CAR therapy targets are generated, it may be possible to test their efficacy and safety in a humanized SCID pigs that are xenografted with a human tumors. Other CAR therapies that are currently under development are CAR-T cells targeting CD20 (102), CD30 (103), CD33 (104), CD7 (105), and CAR-NK cells targeting CD33 (106) and CD19 (107). In addition, as the field of precision medicine continues to grow, a patient's tumor could be xenografted into a SCID pig and a therapy could be tested. Tumors in SCID pigs could be grown to a comparable size to those found in humans and would therefore be a more representative model compared to the limited size of tumors in mouse models. Similar, studies have been performed in hu-PBL-mice, in which interactions between human thyroid tumors and PBLs were studied (108).

### Improving Targeting Imaging Techniques

Pigs are an excellent animal model for surgical and clinical imaging research. Due to their larger size, techniques that are used for humans in the clinics (PET, MRI, CT, US) can also be readily adapted for use in swine.  Pigs with inducible tumors have previously been imaged with CT and MRI, which is proof of concept that these imaging techniques can be performed on pigs (110).

There are also practices that involve targeted imaging of tumors using small peptides and molecules. SCID mice have previously been used for such studies for ovarian (112), nasopharyngeal, breast (113), hepatic (114), lung cancer (115), and others. SCID mice are useful animal models for proof of concept studies that certain molecules and peptides can specifically bind to certain tumor types. After preliminary testing has been completed in mice, SCID pig models engrafted with human cells could then be used for testing these targeting techniques with respective imaging equipment that would be used in the clinics. As an example, human ovarian carcinomas expressing high levels of Claudin 3/4 expression will grow in SCID pigs (45). A Clostridium perfringens enterotoxin (CPE) peptide can specifically bind to Claudin 3/4 (112, 116), and such a SCID pig ovarian cancer model can be used as an imaging and therapeutic target of the CPE peptide in targeting ovarian carcinomas in such a way that it is translatable to human patients.

### Development of Patient Derived Xenograft Models in SCID Pigs for Personalized Drug Testing

Since SCID pigs have previously been shown to accept xenografts of human cancer cells (13), as well as pluripotent stem cells (24, 46), it would be expected that they would also accept solid tumor tissues as well. Patient derived xenograft (PDX) and cell derived xenograft models have previously been utilized in SCID mouse models for patient specific drug testing (117). SCID pig models can also be developed for these purposes. Due to higher similarity in metabolism between humans and pig (32) compared to mice, drug responses in the pig would likely lead to more directly comparable responses to those that would be found in humans (33). Additionally, the size of the pig would also allow representative drug doses to be tested that could be applied to future doses for human patients.

### CONCLUDING REMARKS

Here we have described many of the novel uses of SCID pigs in oncology research involving the use of xenotransplantation of human tumor tissues, HSCs, and lymphoid tissues. The full potential of these animals will be realized when biocontainment facilities are more readily available and survivability of SCID pigs improved. Additionally, dissemination of handling protocols will be essential to prolonging the lives of these animals for long-term studies.

### REFERENCES

Research groups generating SCID pigs are at the forefront of creating a new animal model that can be used for translational preclinical research. We have learned an incredible amount of information by use of small animal mouse models for cancer research. However, in order for therapies to be developed and tested thoroughly, they now need to be evaluated in a larger animal model that better represents human disease states and which can provide realistic opportunities for improved modeling of imaging and surgical approaches. As such, we believe that SCID pig models will provide a foundation for researchers to gain valuable and translational results to improve patient outcomes in a clinical setting.

### AUTHOR CONTRIBUTIONS

AB wrote manuscript and designed figures. JC and CL edited and revised manuscript. CT wrote, edited, and revised manuscript.

### FUNDING

This work was funded by grant 1R24OD019813-03 from the National Institutes of Health.

### ACKNOWLEDGMENTS

We would like to thank Sara Charley and Timothy Egner on insightful discussions of this work.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc. 2018.00559/full#supplementary-material

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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