Bridging the Gap from Bench to Bedside: A Call for In Vivo Preclinical Models to Advance Endometrial Cancer and Cervical Cancer Immuno-Oncology Research

Abstract

Advanced-stage endometrial and cervical cancers are associated with poor outcomes despite contemporary advances in surgical techniques and therapeutics. Recent clinical trial results have led to a shift in the treatment paradigm for both malignancies, where immunotherapy is now incorporated in the upfront setting for most patients with advanced endometrial and cervical cancers as the standard of care. Impressive response rates have been observed, but unfortunately, a subset of patients do not benefit from immunotherapy, and survival remains poor. Continued pre-clinical research and clinical trial development are crucial for our understanding of resistance mechanisms to immunotherapy and maximization of therapeutic efficacy. In this setting, syngeneic models are preferred over xenograft models as they allow for evaluation of the tumor-immune interaction in an immunocompetent host, most closely mimicking the tumor-immune interaction in human cancer patients. Unfortunately, significant disparities exist regarding syngeneic models in gynecologic malignancy, where queries from multiple large bioscience companies confirm no commercial availability of endometrial or cervical cancer syngeneic cell lines. Few published data exist regarding the recent development of several endometrial and cervical cancer syngeneic cell lines, warranting further investigation. Closing the disparity gap for pre-clinical models in endometrial and cervical cancer will support physician-scientists, basic and translational researchers, and clinical trialists who are dedicated to improving outcomes for our patients with advanced disease and poor prognosis.

Introduction

Endometrial (EC) and cervical cancers (CC) affect a significant number of women in the United States, with an estimated incidence of 65,000 and 14,000 cases in 2023, respectively ¹. Unfortunately, despite continued evolution in surgical techniques, radiation therapy, chemotherapy, and targeted therapies, the death rate for EC has continued to rise over the last ten years and has remained stagnant for patients with CC ¹. Advanced and recurrent EC and CC are associated with a poor prognosis, with an estimated five-year overall survival of less than 20% ^2,3.

Recent landmark randomized clinical trials, including NRG-GY018, RUBY, KEYNOTE-826, and KEYNOTE-158, have demonstrated unprecedented improvements in oncologic outcomes when immunotherapy in the form of PD-1 antibody (pembrolizumab in NRG-GY018, KEYNOTE-826, KEYNOTE-158; dostarlimab in RUBY) is added to chemotherapy for selected patients with metastatic or recurrent EC and CC and have changed our standard-of-care ^3–7. Pembrolizumab is an anti-PD1 antibody that first received FDA-approval for recurrent microsatellite instability-high endometrial cancers in 2022, based on data from the phase 2 trial KEYNOTE-158, which demonstrated an overall response rate of 48% in this setting. More recently, in NRG-GY018, a 70% and 46% reduction in risk of recurrence was observed for both mismatch repair deficient (MMRd) and proficient (MMRp) metastatic and recurrent endometrial cancer patients, respectively, with the addition of pembrolizumab to carboplatin and paclitaxel ⁴. Similarly, in the RUBY study, dostarlimab is an anti-PD-1 antiboody that when combined with carboplatin and paclitaxel, was associated with a 36% reduction in disease progression at 24 months compared to carboplatin and paclitaxel alone among all patients with advanced or recurrent endometrial cancer ⁶.

In KEYNOTE-826, pembrolizumab in combination with platinum-based chemotherapy, with or without bevacizumab, was associated with a 38% risk reduction in disease progression in patients with PD-L1 CPS ≥ 1 CC, with recently presented data from The American Society of Clinical Oncology (ASCO) 2023 Annual Meeting demonstrating a significant overall survival (OS) benefit of 28.6 vs. 16.5 months (HR 0.60, p<0.001). ^3,8. As the treatment paradigm for metastatic and recurrent endometrial and cervical cancers changes before our eyes, we must challenge ourselves to answer the question, “What next?”. While pembrolizumab and dostarlimab have demonstrated remarkable clinical efficacy in many patients, significant gaps persist in our understanding of their mechanism of action, strategies to modify outcomes in non-responders, and specific biomarkers of response.

Novel early-phase clinical trials are attempting to improve immunotherapy response in gynecologic cancers by a variety of mechanisms, including combination therapies and adoptive T cell therapy, including Chimeric Antigen Receptor T cells (CAR-T cells) and Tumor-Infiltrating Lymphocyte (TIL) therapy, which have shown promising results in patients with cervical cancer ⁹. Additionally, numerous promising investigations are underway, including a phase III randomized trial of radiation +/− pembrolizumab for newly diagnosed, early stage high intermediate risk MMRd EC (NCT04214067) and a phase II trial of nivolumab with or without ipilimumab in MMRd recurrent EC (NCT05112601) which seek to further transform the treatment landscape for patients with newly diagnosed and recurrent EC ¹⁰. These clinical trials utilizing combination therapy, targeted agents, and the improved molecular classification of gynecologic cancers offer promise to broaden the framework of treatment for clinicians and improve therapeutic options for upfront and recurrent EC and CC.

The importance of syngeneic models to pre-clinical study and gynecologic under-representation in commercial availability

The importance of early drug development and novel clinical trials designed to maximize the efficacy of immunotherapy is paramount to further improve outcomes for cancer patients. Advancement in early-phase studies depends heavily upon the groundwork laid by pre-clinical investigation. In-vitro and in-vivo studies, often utilizing pre-clinical mouse models, are necessary for drug design, mechanistic exploration, and clinical trial development ¹¹. Specifically, requirements to submit a clinical trial letter of intent (LOI) to the National Cancer Institute (NCI) via the Experimental Therapeutics Clinical Trials Network (ECTCN) or Cancer Therapy Evaluation Program (CTEP) mechanisms include that the drug combination must be evaluated in two disease-relevant cell lines and two disease-relevant xenograft animal models ¹².

Mice are the most utilized animal model for studying human diseases, as they have biological similarities to humans, an accelerated life span with one mouse year equaling 30 human years, and can be inbred and undergo genetic manipulation to facilitate accuracy and replicability in experimental design ^13–15. Syngeneic mouse models utilize genetically identical mouse tumor cells grown in an immune-competent mouse, allowing researchers to investigate tumor-immune interactions and evaluate the efficacy of immunotherapy interventions ^13,16. In contrast, xenograft models utilize cells from different species and must be inoculated into immunocompromised mice, which therefore limits the study of interactions between the tumor and the host immune system.

Syngeneic mouse models rely on the engraftment of established same-species tumor models instead of spontaneous generation, which allows for low cost and reproducibility in data. However, the non-human origin of the cell lines limits the applicability to human studies through inherent differences in the tumor microenvironment, tumor growth kinetics and biology, and molecular markers and genomic mutations between murine and human cancers ¹⁷. Prior studies have demonstrated that syngeneic models have displayed decreased intra-tumoral heterogeneity, decreased T-lymphocyte infiltration, diverse immune cell infiltration patterns, and a more mesenchymal-like phenotype compared to human cancer tumors ¹⁷. Accordingly, these differences emphasize that commonly utilized mouse syngeneic models derived from a particular tissue may only partially recapitulate the properties of human tumor populations of the same tissue. As a result, translating findings from mice to humans solely based on the tissue of origin is complex and not without limitation. Additionally, resistance to checkpoint inhibitors may arise through various mechanisms, including through activation of the β-catenin pathway and epigenetic silencing ^18,19.

In contrast, xenograft models utilize cells from different species and must be inoculated into immunocompromised mice ^16,20. Traditional cell line-derived xenograft (CDX) models are commonly used in preclinical cancer research. However, these models are limited in their heterogeneity and representation of the microenvironment within a tumor and the limited host immune system limits the study of the immune system and immunotherapy. Moreover, long-term culture of cancer cell lines in vitro can introduce genetic drift and epigenetic modifications through multiple passages, which may influence experimental findings. In contrast, patient-derived xenograft (PDX) mouse models are developed through the implantation of patient-derived tumor samples into immunodeficient mice ^13,15. Therefore, these models more adequately preserve the histology and molecular features of tumors and therefore, may have a more similar response to treatment as in human tumors. The development of robust, clinically relevant PDX models has logistical challenges, including access to viable human samples, challenges with consistent tumor engraftment, and often lengthy time for tumor development ^13,15.

Humanized mouse models that utilize immunodeficient mice co-engrafted with human immune cells and cancers hold promise but have logistical challenges, are often cost-prohibitive, and have limited capacity to truly recapitulate the complexity of the native human or mouse immune system ¹³. Humanized mouse models that utilize immunodeficient mice co-engrafted with human immune and cancer cells hold promise but have limitations, including high cost and a limited capacity to truly recapitulate the complexity of the native human or mouse immune system and tumor microenvironment ¹³. To overcome these limitations, researchers have incorporated healthy donor peripheral blood mononuclear cells (PBMCs) and hematopoietic stem cells into xenograft and patient-derived xenograft (PDX) models. Utilizing PBMCs to create a humanized mouse model has several advantages, including rapid engraftment and enrichment for human CD3+ T-cells, which is beneficial for understanding T cell-mediated tumor regression. However, many existing humanized cancer mouse models may exhibit allogeneic responses between the implanted human tumors and injected immune cells and do not provide an autologous adaptive immune response against patient-specific tumor antigens²¹.

Lastly, in-vitro model systems have value as they allow for genomic manipulation, high-throughput screening, and efficient drug sensitivity testing. In CC and EC, several patient-specific pre-clinical model systems have been described, including spheroids, patient-derived organoids, and organ-on-chip systems, which provide a more accurate representation of the tumor microenvironment and maintain the phenotype and heterogeneity of tumors more closely than traditional two-dimensional cell culture ^22–24. However, despite promising early data with tumor organoids, they are limited in their consistency and further research is needed to optimize these systems at large scale.

Therefore, to advance research toward the discovery of novel immunotherapy agents and strategies to improve immunotherapy outcomes in EC and CC patients, syngeneic models are required as they allow for study of the interactions between the tumor and the host immune system and have reproducibility and are cost-effective foe researchers compared to other strategies. Currently, there are no commercially available syngeneic models of EC or CC for investigator use. On review of three leading biosciences companies as of June 2023, of 142 commercially available syngeneic lines, gynecologic cancers represent the minority of available products (2.8%), with four ovarian cancer lines available, and no EC or CC cell lines ^25,26. In contrast, breast cancer models represent 20.4% (n=29), hematologic malignancy models 12.7% (n=18), and colon cancer models 7.7% (n=11). It is important to note the creation of syngeneic models for EC and CC is a challenging task. Recently, a large-scale, publicly accessible resource of syngeneic mouse models named Tumor Immune Syngeneic Mouse (TISMO) has been created. TISMO includes 68 syngeneic mouse tumor models across 19 cancer types, of which 832 were from immune checkpoint blockade studies²⁷. In lung cancer, there are only two commercially available C57BL/6 derived murine lung tumor lines capable of forming orthotopic lung tumors in immunocompetent hosts²⁸. Within mammary carcinoma of the breast, the 4T1 syngeneic cell line has been utilized in a variety of experiments involving anti-PD1 and anti-CTLA4 immunotherapies ^29,30

Existing syngeneic models in endometrial and cervical cancer

There is limited published data regarding syngeneic EC models that are not commercially available ^31–35. Fedorko and colleagues characterized the MECPK cell line derived from primary endometrial tumors of Pgr^cre/+Pten^f/fKras^G12D mice ³². In-vivo experiments yielded uterine tumor development in 59% of mice within one month of injection with MECPK cells. Notably, graft establishment for the model requires surgery with ovariectomy, uterine abrasion with direct MECPK cell injection into the uterine lumen, and exogenous estrogen. While the authors discuss that this model best resembles a type I endometrioid cancer, H&E staining is more comparable with a high-grade histology, which is inconsistent with the majority of copy-number low endometrial tumors in The Cancer Genome Atlas ³⁶. Further, mismatch repair proteins were reported as intact, and few tumor-infiltrating lymphocytes were noted, consistent with a microsatellite-stable phenotype ³². In this model, tumors exhibited loss of PTEN, activation of K-Ras, PI3K, and MAPK pathway activation. These tumors possessed established genetic defects and established drivers of human endometrioid endometrial cancer ²⁰.

An additional syngeneic model utilizing an LKB1^fl/fl/p53^fl/fl genetically engineered mouse has been described by Bae-Jump and colleagues ^31,33–35. For this model, LKB1 and p53 are inactivated via injection of the AdCre virus into the uterine horns, with the development of endometrial cancer approximately eight weeks following inoculation. For this model, LKB1^loxP/p53^loxP and LKB1^loxP/p53^loxP mice were intercrossed through multiple generations to create colonies of homozygous LKB1^fl/fl/p53^fl/fl double conditional knockout mice. This model has been utilized to evaluate the complex interplay of metabolic syndrome, metformin, tumor progression, and angiogenic pathways. Neither syngeneic model described above has been described in currently published immunotherapy studies.

Similarly, there are limited published reports of CC syngeneic mouse models. Limitations in previously reported CC models included inconsistent and slow-growth kinetics, lack of high-grade dysplasia precursors to mimic CC carcinogenesis, and inability to recapitulate clinically relevant responses ^37–39. Henkle and colleagues reported the development of a spontaneous high-risk HPVE6/E7-expressing carcinoma cell line, Tal3. This was accomplished through injection of AME-16 plasmids encoded with HPV16 or 18 E6/E7 luciferase, AKT, c-myc, and Sleeping Beauty transposase into the cervicovaginal tract of C57BL/6 mice that were electroporated to introduce DNA into the cells ⁴⁰. Subsequently, utilizing an intraperitoneal metastasis from a cervicovaginal AMES-16-derived tumor, cells were subcutaneously injected into C57BL/6 mice, and 100% tumor penetration was demonstrated, which led to the development of the Tal3 cell line. These tumors are either well- or poorly-differentiated squamous cell carcinomas and express HPV-oncogene ⁴⁰. Further, they demonstrate robust tumor-infiltrating lymphocyte presence and have strong expression of T cell exhaustion markers, including PD-1 and CTLA4. These mentioned preclinical models and cell lines represent groundbreaking advancements in the field of gynecologic oncology and also demonstrate the possibility of generating additional preclinical EC and CC platforms to evaluate the efficacy and safety of immunotherapies or other treatment modalities.

Conclusions: syngeneic models’ development will lead to improved endometrial and cervical cancer outcomes

Syngeneic models of CC and EC are necessary to advance treatment outcomes in patients with EC and CC, and this barrier has the potential to impact the future of patients with EC and CC globally. While pembrolizumab and dostarlimab have shown substantial benefits in the treatment of EC and CC, considerable knowledge gaps exist, including understanding mechanisms of resistance and strategies to overcome it.

Intrinsic resistance to immune checkpoint inhibitor therapy in gynecologic cancers is poorly understood. EC and CC have significant heterogeneity with regard to tumor biology, patterns of metastasis, histology, and molecular features, which dramatically impacts prognosis and response. In both malignancies, it is well understood that cell populations of the innate and adaptive immune systems are predictive of outcomes, with T-cell infiltration associated with improved response and survival, but the role of B cells is less defined ^9,10,41. Several promising approaches to improve immunotherapy response and combat resistance have been described, including reprogramming an immunosuppressive tumor microenvironment via rationalized combination therapies and microbiome-directed interventions. Still, our capacity to explore these interventions is limited without applicable preclinical models. However, there are important, often immeasurable, factors that may not easily translate to pre-clinical models, including racial differences, ethnic disparities, diet, body composition, and environmental stressors. Continuous, collaborative efforts must be made to bridge the gap between pre-clinical cancer models and our patients.

The lack of commercially available syngeneic mouse models may also negatively impact early-career gynecologic oncologists seeking physician-scientist and clinical-trialist careers who are unlikely to have the knowledge or funding to develop their own mouse models and be unable to generate necessary preliminary data to submit trial concepts to the NCI or industry partners ¹¹. Prior data supports that gynecologic cancers have significant funding disparities relative to other malignancies ⁴². Specifically, in an analysis of NCI funding patterns from 2007–2014, breast cancer received an average of $1.8 million per person-years of life lost, compared to $87,000 for CC and $57,000 for EC ⁴². Therefore, without efforts to advance pre-clinical models EC and CC, these funding disparities and their negative impact on patient outcomes are poised to prevail.

Collaborative efforts between translational and basic science researchers, clinicians, funding agencies, and industry partners are crucial to prioritize and support the creation of syngeneic models for gynecologic cancers. By expanding available syngeneic mouse models for EC and CC, our specialty will possess additional tools to understand tumor-immune interactions and mechanisms of resistance further, evaluate novel immunotherapy strategies, and advance treatment outcomes for patients.

Statement of translational relevance.

Clinical trial development requires pre-clinical data, often including drug evaluation in two disease-relevant cell lines as well as animal models. With the evolving treatment landscapes for endometrial and cervical cancers, the incorporation of immunotherapy is becoming the standard of care for many patients. Pre-clinical data in this setting is best served by syngeneic mouse models, which allow for investigators to evaluate interactions most accurately between the tumor, tumor microenvironment, and immune system. Unfortunately, gynecologic malignancies are significantly under-represented regarding commercially available syngeneic models, with none existing for endometrial or cervical cell lines. Development and improved availability of such models are needed for clinical trial design and advancement of therapies for two gynecologic malignancies where advanced stage disease is still associated with poor survival.