Age and Sex Differences in the Eμ-TCL1 Adoptive Transfer Mouse Model of CLL

<jats:title>Abstract</jats:title>         <jats:p><jats:italic>Background:</jats:italic>The Eμ-TCL1 syngeneic model is the most widely used mouse model of chronic lymphocytic leukemia and has been extensively used to understand the pathogenesis of select genes, the effect of the immune microenvironment and for pre-clinical drug development studies. Recently there has been an increasing awareness of the impact of age and sex differences on not only the development of cancers but also the efficacy and toxicity of specific cancer therapies. However, despite the predominance of older males in CLL patient demographics, the Eμ-TCL1 adoptive transfer studies have used almost exclusively young female recipient mice. <jats:italic>Methods:</jats:italic>In this study we performed primary and secondary adoptive transfer experiments in order to understand the impact of recipient age and sex on the development of disease as assessed by flow cytometry and survival in the Eμ-TCL1 adaptive transfer model. <jats:italic>Results:</jats:italic>We found that young female recipients had pro-longed survival in a primary adoptive transfer, however sex differences were not observed in a subsequent secondary adoptive transfer experiment. Furthermore, while recipient age did not have a significant effect on the rate of disease establishment or survival in female mice, aged male mice had a significantly decreased rate of Eμ-TCL1 tumor engraftment. <jats:italic>Conclusions:</jats:italic>These findings suggest that age and sex differences must be considered in the experimental design of studies using the Eμ-TCL1 adaptive transfer model of CLL.</jats:p>

section_content: Age and sex are well established as signi cant variables in both the incidence of cancer and the overall survival of patients [1] [2] [3] [4] [5]. As with many other cancers, the incidence of chronic lymphocytic leukemia (CLL) increases with age and the median age at diagnosis is 70 years. Additionally, CLL also shows a signi cant sex bias toward males by nearly 2:1 [6]. Furthermore, in CLL patients, both age and sex have been shown to be important prognostic indicators, so much so that age is included in the CLL international prognostic index [7] and patient sex has been shown to be a signi cant variable in overall survival, treatment response, and therapeutic toxicity [8]. 

Importantly, in the age of immunotherapy, both age and sex have also been shown to have profound effects on the immune system [9, 10]. Indeed, the cardinal features of age related immunosenescence: impaired ability to respond to new antigens, unsustained memory responses, greater propensity for autoimmune responses, and lingering low-grade in ammation have all been implicated in the failure of cancer control [11] [12] [13]. Furthermore, there have been many studies showing signi cant sex differences in pharmacokinetics [14] and the adverse effects of cancer treatment [15, 16]. Despite recognition of the importance of age and sex differences in human cancer, the pre-clinical models used to understand cancer pathogenesis and the development of novel therapies have often ignored potential age or sex differences. 

Murine models are an important tool in both drug development as well as in understanding basic disease biology and pathogenesis. First published in 2002, the Eμ-TCL1 model in which the human T cell leukemia 1 (TCL1) oncogene is expressed under the control of the V H -promoter and Ig H -Eµ-enhancer, has become a mainstay of CLL pre-clinical research [17]. While by no means the only murine CLL model [18], the Eμ-TCL1 model recapitulates many of the feature of human CLL including the canonical expansion of a CD5+ B cell population, the indolent disease course, and even the profound immune dysfunction observed in patients. In particular, studies aimed at understanding the immune microenvironment often necessitate the use of immunocompetent murine models such as the Eμ-TCL1 model [19]. Given prior reports of sex differences in the Eμ-TCL1 model [20] and the preponderance of evidence suggesting sex differences are evident in other syngeneic models [21] [22] [23] [24] we sought to perform a prospective analysis of the effect of sex and aging on the Eμ-TCL1 adoptive transfer model. 

Herein we report that young female recipients had prolonged survival compared with male counterparts in a primary engraftment. This effect was most prominent in more indolent disease presentations and were not present in a more aggressive secondary engraftment. Furthermore, while recipient age did not have a signi cant impact on survival in female mice, aged male mice had a signi cantly increased rates of failure to engraft and slower rates of tumor establishment compared to younger recipients. These ndings suggest that while both male and female mice can and should be included in pre-clinical studies using the Eμ-TCL1 model, age and sex differences must be adequately controlled for and considered in the interpretation.
section_name: Background

section_name: Methods

section_content: All animal experiments were performed under protocols approved by The Ohio State University Institutional Laboratory Animal Care and Use Committee. Development of B cell malignancies in the Eμ-TCL1 and Eμ-Myc transgenic mice have been previously described [17, 25]. 

7-week-old C57BL/6 recipient mice were purchased from Jackson laboratories (Strain # 000664). 

Humanized cereblon mice (hmCRBN) [26] were a gift from Benjamin Ebert (Harvard Medical School) and were bred and aged in-house at Ohio State University. hmCRBN were >98% C57Bl/6 by SNP testing (Charles River Laboratories).
section_name: Mouse Models

section_content: Donor mice were assessed by ow cytometry of peripheral blood, moribund homozygous Eμ-TCL1 mice with >75% CD19+CD5+ peripheral blood disease burden and enlarged spleens were euthanized by CO 2 inhalation, splenocytes isolated by mechanical dissociation and viably cryopreserved prior to use in adoptive transfer experiments. Recipient mice were injected via the tail vein with 10 7 viable splenocytes (>95% CD19+CD5+ by ow cytometry) in sterile Dulbecco's phosphate-buffered saline (DPBS). Survival endpoint was determined using prede ned euthanasia criteria including body condition score [27], lethargy, spleen size greater than one third the length of abdomen, or loss of >20% body weight. In nontreatment studies, mice were monitored twice weekly for weight loss and other early removal criteria. In aging studies, mice received daily i. p. injections of vehicle (5% DMSO in sterile DPBS) prepared daily. Mice receiving active drug were not included in this analysis. In all studies, fortnightly cheek bleeds were performed to assess tumor burden. Survival analysis excluded mice who failed to engraft, de ned as not reaching >15% CD19+CD5+ in the peripheral blood over the study period.
section_name: Adoptive transfer experiments

section_content: Peripheral blood was collected in EDTA microtainers via submandibular bleed and was stained using a lyse no wash protocol. Brie y 20 μL whole blood was blocked using 1 μL Mouse BD Fc Block (BD) then incubated at room temperature for 15 minutes with uorochrome conjugated antibodies according to 3. Results
section_name: Flow Cytometry

section_content: Despite the male sex bias in many areas of research [28], work in the Eµ-TCL1 model has almost exclusively relied upon young female mice. In the last 4 years 32 papers reported Eµ-TCL1 adoptive transfer experiments. Most studies were performed using 6-12-week-old recipient mice with only 1 paper reported using mice older than 4 months [29]. Furthermore, of the 32 papers only 2 reported using male mice, but the data was not analyzed by sex. 11 of the papers did not report the age of the recipient mice and 20 did not report the recipient sex (Table S1 ). 

We therefore rst sought to understand the effect of donor and recipient sex on the establishment of Eµ-TCL1 adoptive transfer (AT) tumors. 8-week-old male and female C57Bl/6 mice received an adoptive transfer from one of ve donors (Table 2 ). Establishment of the Eµ-TCL1 tumor was measured by periodic assessment of CD19 + CD5 + leukemia cells in the peripheral blood, representative ow plots are presented in Fig. 1a. Forty ve of the 48 mice (94%) reached the threshold for engraftment (> 15% CD19 + CD5 + cells in peripheral blood). At 3 months post-AT sex-mismatched recipients, either male donor into female recipient (mean = 75 x10 3 cells per µL) or female donor into male recipient (mean = 56 x 10 3 ) had signi cantly higher leukemia cell counts than sex-matched recipients (matched females mean = 8. 5 x10 3 and matched males = 26 x10 3 ) (Fig. 1b ). As expected, peripheral blood disease burden at 3 months was strongly negatively correlated with survival (Fig. 1d ). 1 PB ow performed in the week prior to tissue collection. 

We next assessed the impact of donor and recipient sex on overall survival. Donor sex had no impact on the survival suggesting that there is not a cell intrinsic sex difference in the propensity to establish a leukemia (Fig. 2a ). In contrast, recipient sex signi cantly affected overall survival with female recipients having prolonged survival as compared to male recipients (Fig. 2b ). Interestingly, while sex mismatch signi cantly increased the rate of disease establishment (Fig. 1 ), it did not result in a signi cant difference in survival (Fig. 2c ). These nding are in contrast to prior reports which suggested that male mice had a survival advantage in the Eµ-TCL1 model [20]. However, prolonged survival of female recipients was also observed in the Eµ-Myc model, another syngeneic model of B cell malignancy (Figure S2 ). Further, this data suggested that the sex difference was more prominent in female recipients with longer survival. We therefore sought to understand the effect of donor variability on the observed sex differences. Of the donors, 2 had a more aggressive phenotype with a median survival of 87 days while 3 were more indolent with a median survival of 140 days (Fig. 2d ). Recipients of "aggressive" donors showed no sex difference in survival whereas in more "indolent" donors, the sex difference was more apparent (Fig. 2e-f ).
section_name: Sex differences in Primary engraftment

section_content: It has been previously established that passaging Eµ-TCL1 cells in a mouse prior to use in a subsequent experiment allows both for acceleration of the disease course in subsequent studies [20] as well as allowing for the expansion of primary donor cells and therefore limiting the impact of donor variability on subsequent engraftments. We therefore sought to understand if the observed sex differences were maintained in a secondary engraftment. Splenocytes were collected from recipients in the primary engraftment study (P1) and were then adoptively transferred into secondary recipients (P2). Eight donors were selected from the primary experiment including male and female P1 donors and P1 donors from both the aggressive and indolent groups. P2 donor characteristics are outlined in Table 3. As expected, neither donor nor recipient sex signi cantly affected the establishment or survival of P2 recipients (Fig. 3a-c ). Interestingly, while there was no signi cant difference in the rate of tumor establishment in recipient of cell from "aggressive" and "indolent" P1 donors, there remained a signi cant difference in the survival of P2 recipients, with median survival of 56 and 65 days respectively (Fig. 3d ).
section_name: Sex differences in secondary engraftment

section_content: Given the lack of sex differences in secondary engraftment, we proceeded with a secondary engraftment for the subsequent investigation of the impact of recipient age on survival. Cells from a single P1 recipient (F2017, male, survival 84 days, > 96% CD19 + CD5 + in PB and spleen) was used to engraft three age cohorts of hmCRBN recipient mice: young (3-6 months), middle aged (9-12 months), and old (18-22 months). Upon establishment of tumor (> 15% CD19 + CD5 + cells in PB) mice were treated with daily intraperitoneal injections of vehicle (5% DMSO, in sterile water) as part of a parallel study not included in this manuscript. 

In female recipients, recipient age had no effect on the rate of Eµ-TCL1 tumor establishment (median 15 days for all groups, Fig. 4a, c ) or overall survival (median 49, 56, and 48 days respectively, Fig. 4e ). Conversely, in male recipients, there was an age-related decrease in the rate of Eµ-TCL1 tumor establishment (Fig. 4b ). A time to event analysis in which engraftment was de ned as reaching greater than 15% CD19 + CD5 + cells in the peripheral blood showed that old mice had signi cantly delayed engraftment than younger recipients (median 50 days c. f. 22 and 36, Fig. 4d ). Furthermore, in the male mice, the older cohorts had higher rate of failure to engraft than younger recipients (0% failure in young c. f. 41% in old cohort). Interestingly, when considering only male recipients that met engraftment criteria, no signi cant effect on survival was observed. Indeed, old male recipients had a trend to slightly shorter overall survival than younger counterparts (Fig. 4f ). However, this observation is confounded by the high rate of failure to engraft in the old cohort. The median survival of both the male and female mice was 55 days when all age cohorts were included.
section_name: Age impacts secondary engraftment of male recipients

section_content: Here we report signi cant effects of recipient age and sex on recipient survival in immunocompetent syngeneic Eµ-TCL1 mouse model of CLL. Female recipients of adoptive transfer had signi cantly prolonged survival as compared to male recipients, an effect most pronounced in primary adoptive transfer and when more indolent donors were used. Conversely, no sex differences were observed in secondary adoptive transfer experiments. Subsequent studies were performed to investigate the impact of recipient age on disease progression and found that while age was not a signi cant factor in the survival of female mice, in male mice older recipients had signi cantly slower engraftment but shorter survival than young mice. 

Mouse models are an incredibly powerful tool to both further our understanding of cancer biology and in developing new therapeutics. Indeed, in vivo mouse studies are often an essential part of pre-clinical drug development. However, they can fail to accurately recapitulate the variability of responses seen in humans. There are a multitude of factors that contribute to the representativeness of a model, including the use of immunocompetent syngeneic mouse models vs immune-incompetent mice and human derived cells, inbred vs outbred mouse stains, or the use of speci c pathogen free vs 'dirty' mice. While the aforementioned factors are often determined by model availability, age and sex difference can still be investigated. These kinds of studies are often resisted on the basis of feasibility concerns, namely that young female mice are generally cheaper, can be re-housed in long survival studies and are less aggressive than males. Finally, many researchers cite the additional cost incurred to su ciently power experiments for the analysis sex differences [30]. Finaly, published data becomes precedent leading to entrenchment of established protocols. However, the use of age and sex diverse mice may provide additional useful information, particularly as the roles of sex differences, aging, and senescence have an accepted contribution to the development and immune response to cancer. The ndings of this study support the conclusion that both male and female recipients can be used as recipients in the Eµ-TCL1 models. Furthermore, age diverse female recipients may also be used, while greater caution must be applied in the use of male recipients older than 12 months. 

No sex difference was observed between male and female donors in this study. However, the number of donors was small, and a larger study would be required to more systematically understand the contribution of sex to donor cell engraftment. Interestingly our ndings of a female survival advantage in adoptive transfer experiments are in contrast with prior work by Koch et al, which showed a signi cant survival advantage for male transgenic mice and in male recipients of both primary and secondary engraftment. Interestingly, our ndings are in line with the human data in which female CLL patients have greater survival as compared to male patients [8]. We further observed the same pattern in a second syngeneic model of B cell malignancy. Our study and the Koch study also took different approaches to donor variability, where Koch et. al. used only sex matched adoptive transfer in which larger numbers of donor mice (n = 11 each male and female donors) were included, our approach was to use fewer donors engrafted into larger number of recipients of both sexes. We were therefore able to analyze the impact of donor to donor variability on the presentation of sex differences in this model. It is notable that there was a divergence in the rate of disease establishment, in which sex mismatched engraftments has signi cantly greater peripheral blood disease burden, and the overall survival of the mice. This suggests that there may be sex differences in the distribution of disease that the present study was not able to adequately address. Further studies are required to understand how sex differences may alter disease distribution in recipient mice. 

In addition to the impact of sex, we also analyzed the impact of recipient age in the Eµ-TCL1 model. In female mice there was no signi cant difference in the survival of young vs old mice whereas in male recipients, the old cohort (18-22 months at AT) had higher rates of tumor rejection and slower engraftment. However, amongst male recipients who reached the engraftment criteria, there was so signi cant difference in the overall survival. This apparent contradiction may be due to the shorter expected lifespan of male mice than female mice [31]. While there is no direct comparison of mouse and human age, the best approximation suggests that mice reach sexual maturity at 8-12 weeks of age and female mice enter reproductive senescence at 10-15 months, which correlates to a human age of approximately 50 years. This suggests that 6-12-week-old mice, which are most used in pre-clinical studies, are unlikely to be representative of the immune system present in CLL patients who are generally over the age of 65. However, a signi cant limitation of the present study is that it is only an observational study and does not provide insights into the mechanism of age or sex differences. Further research is required to fully elucidate the mechanisms responsible for the observed age and sex differences.
section_name: Discussion

section_content: In this study we demonstrate that while the Eµ-TCL1 model can be adoptively transferred into male and female mice regardless of age, care must be taken to appropriately control for differences in engraftment and survival. While young female recipients of primary adoptive transfer had signi cantly longer survival than males, these differences can be diminished by secondary passage of cells. These nding should encourage the use of both age and sex diverse studies using the Eµ-TCL1 mouse model.
section_name: Conclusions

section_name: Acknowledgments

section_content: We would like to thank Drs. Benjamin Ebert and Emma Fink for providing the humanized cereblon mice and Dr. Carlo Croce for providing the Eμ-TCL1 mouse.
section_name: Acknowledgments

section_name: Availability of data and materials

section_content: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request
section_name: Availability of data and materials

section_content: The authors declare no con ict of interest Funding D Warren Brown Foundation, R35 CA197734, ACS RSG-19-151-01-LIB
section_name: Competing interests

section_name: Supplementary Files

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SupplementalAgeandSexDifferencesintheEuTCL1adoptivetransfermousemodelofCLL. docx
section_name: Supplementary Files