Various malignancies are reproducibly cured in mouse models, but most cancer immunotherapies show objective responses in a fraction of treated patients. One reason for this disconnect may be the use of young, lean mice lacking immune-altering comorbidities present in cancer patients. Although many cancer patients are overweight or obese, the effect of obesity on antitumor immunity is understudied in preclinical tumor models. We examined the effect of obesity on two immunotherapeutic models: systemic anti–CTLA-4 mAb and intratumoral delivery of a TRAIL-encoding adenovirus plus CpG. Both therapies were effective in lean mice, but neither provided a survival benefit to diet-induced obese BALB/c mice. Interestingly, tumor-bearing leptin-deficient (ob/ob) obese BALB/c mice did respond to treatment. Moreover, reducing systemic leptin with soluble leptin receptor:Fc restored the antitumor response in diet-induced obese mice. These data demonstrate the potential of targeting leptin to improve tumor immunotherapy when immune-modulating comorbidities are present.
Obese humans face an array of health issues, including an increased risk for cancer. Cancer-associated morbidity and mortality are also greater in obese persons. The reason for this obesity–cancer link is multifactorial, but generalized immune system dysfunction is a significant contributor (1). Increased cancer risk during obesity has also been ascribed to elevated production of tumor-promoting hormones and growth factors by adipocytes. As weight increases and develops into obesity, visceral adipose tissue actively contributes to a state of systemic, low-level inflammation. Growing evidence suggests chronic inflammation, resulting from obesity and/or tumor growth, plays a salient role in tumor cell survival/proliferation and immune suppression (2–4). The obesity epidemic makes understanding how this common comorbidity influences the immune response to cancer imperative (5). This is especially true for cancers that correlate highly with obesity, such as renal cell carcinoma (RCC), which affects >60,000 and kills >14,000 people in the United States annually, making it the second-most lethal urologic cancer (6–9).
In this study, we describe the impact of obesity on two immunotherapy strategies. We have previously described an experimental therapy using a recombinant adenovirus encoding TRAIL in combination with a TLR agonist (CpG) in an orthotopic mouse model of RCC (10–12). Delivered directly into the primary tumor-bearing kidney, Ad5-TRAIL/CpG immunotherapy stimulates an abscopal CD8+ T cell–mediated antitumor response that significantly enhances survival in lean but not diet-induced obese (DIO) mice (10, 13). These data implied that some obesity-related factor was responsible for immunotherapy failure. The present study extends our previous work by evaluating immunotherapeutic activity in a second model of obesity caused by the genetic loss of leptin using leptin-deficient (ob/ob) mice, which spontaneously become obese without dietary intervention (14), as well as using a systemically administered checkpoint inhibitor (anti–CTLA-4 mAb). Tumor growth was blunted and overall survival enhanced in lean and ob/ob obese mice after local or systemic antitumor immunotherapy, whereas DIO mice did not demonstrate any benefit with either treatment. The different therapeutic responses between diet-induced and ob/ob obesity led us to posit the elevated leptin in DIO mice was a contributor to therapeutic failure. Indeed, reducing the amount of leptin in DIO mice by systemic delivery of recombinant leptin receptor (ObR):Fc (ObR:Fc) restored immunotherapy efficacy. These data collectively indicate the importance of incorporating clinically relevant comorbidities into preclinical tumor models to broaden the testing of immunotherapeutic protocols. Moreover, our data have identified leptin as a potential therapeutic target for neutralization to enhance immunotherapy efficacy in obese cancer patients.
Materials and Methods
Animals and diets
Female BALB/c mice 7–8 wk old (Charles River Laboratories) were given standard chow or high-fat feed (HFF) (Research Diets; no. 12492) for 20 wk ad libitum. Mice in the HFF group whose body weight was ≥3 SD above the mean weight of the standard chow group were defined as DIO, whereas mice whose body weight was <3 SD above the mean weight of the standard chow group were defined as obese resistant (10). BALB/c mice with a heterozygous deletion of the leptin gene (ob/+) were bred to produce ob/ob offspring. Male and female littermates (ob/+ or +/+) were used as controls. ob/ob and littermate controls were fed standard chow from weaning throughout the duration of experiments. Body composition analysis was performed using an EchoMRI 3-in-1 (Echo Medical Systems, Houston, TX).
Leptin quantitation and neutralization
Serum leptin was measured by ELISA (Chrystal Chem, Downers Grove, IL). Soluble mouse leptin receptor:Fc (ObR:Fc) (15) was purchased from R&D Systems (Minneapolis, MN).
The murine renal adenocarcinoma cell line Renca (CRL-2947; American Type Culture Collection) was transfected with the Sleeping Beauty transposon system to confer stable expression of firefly luciferase and maintained in RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, 1× nonessential amino acids, 50 μM 2-ME, 10 mM HEPES, and 0.05 μg/ml puromycin. Intrarenal (IR) tumor challenge was performed as described previously (10–12). On day 7, mice were injected in the tumor-bearing kidney with sterile PBS or Ad5-TRAIL (109 PFU) (16) and CpG ODN 1826 (5′-TCCATGACGTTCCTGACGTT-3′, 100 μg; Integrated DNA Technologies, Coralville, IA). Anti–CTLA4 mAb or control Ig (10 mg/kg i.p., clones 9D9 and MCP-11, respectively; Bio X Cell, West Lebanon, NH) was given on day 4, 8, 11, 15, and 18 after s.c. tumor challenge (17).
Dendritic cell activation
Splenocytes were cultured for 24 h in RPMI 1640 medium alone or with CpG (10 μg/ml) and then stained for CD8α dendritic cell (DC) (I-Ad+CD11c+CD8α+ cells) or plasmacytoid DC (pDC; I-Ad+CD11c+B220+CD317+ cells) and analyzed by flow cytometry to determine costimulatory molecule expression.
To identify the CD8+ T cells among the CD45.2+ leukocytes located in the tumor-bearing kidney tissue, intravascular staining was done using anti–CD45.2-PE (BioLegend, San Diego, CA) (18). Tumor-bearing kidneys were harvested, manually disrupted, and digested with DNase I (15 μg/ml; Sigma-Aldrich) and Liberase Blendzyme 3 (0.026 Wunsch units/ml; Roche Diagnostics). After blocking with anti-CD16/32 in normal mouse serum, cells were stained (anti–CD45.2-BV650, CD3-v500, CD4-AF700, CD8-BUV395; eBioscience, San Diego, CA, or BioLegend) and analyzed using multiparameter flow cytometry on a BD LSRFortessa (BD Biosciences, San Diego, CA) and FlowJo software (TreeStar, Ashland, OR).
Statistical comparisons of body weight composition, serum leptin, mean fluorescence intensity, and tumor-infiltrating CD8+ T cells were performed using the Student t test. Survival data were compared by log-rank (Mantel–Cox) analysis. All tests were performed using Prism 7 (GraphPad Software). The p values <0.05 were considered statistically significant.
Results and Discussion
Diet-induced versus genetic models of obesity in mice
Murine obesity can be modeled using a diet of HFF or ob/ob. Slowly progressing and clinically relevant DIO using HFF is marked by increased leptin production and systemic inflammation (19–21). Consistent with our previous data (10), ∼50% of BALB/c mice on HFF for 20 wk became obese (Fig. 1A). In contrast, all ob/ob BALB/c mice became obese relative to ob/+ littermates. Furthermore, body composition analysis demonstrated that the elevated body weight resulted in increased body fat in the DIO and ob/ob mice compared with their lean counterparts (Fig. 1B). Serum leptin was expectedly increased in DIO mice compared with mice fed standard chow, and no leptin was detected in the serum of ob/ob mice (Fig. 1C). The serum leptin levels in the lean and DIO mice are consistent with amounts seen in lean and obese humans (22). These data define the starting parameters of the lean and obese (DIO and ob/ob) mice.
Immunotherapy is effective in lean and ob/ob obese mice but not DIO mice
Intratumoral delivery of Ad5-TRAIL/CpG in an orthotopic mouse model of RCC (10, 13, 16, 23) resulted in a significant survival advantage compared with PBS treatment in lean mice, but no difference in survival was noted among PBS- and Ad5-TRAIL/CpG–treated DIO mice (Fig. 2A). In contrast, tumor-bearing ob/ob mice displayed increased survival after Ad5-TRAIL/CpG therapy, paralleling the response seen in the ob/+ lean littermates (Fig. 2B). Lean, ob/+, and ob/ob mice bearing s.c. Renca tumors also demonstrated enhanced overall survival when given systemic anti–CTLA-4 mAb therapy (17), whereas DIO mice failed to respond (Fig. 2C, 2D). Importantly, both Ad5-TRAIL/CpG and anti–CTLA-4 mAb therapies significantly increased survival in HFF obese-resistant mice cohoused with HFF DIO mice (Supplemental Fig. 1), which provides an important internal control to exclude such variables as an effect of diet or microbiome differences as contributing factors. These data suggest obesity alone is not enough to alter the efficacy of immunotherapeutic treatment of tumor-bearing mice.
An effective antitumor immune response requires proper Ag presentation by APCs to CD8+ T cells. Dysfunctional APC would result in poor CD8+ T cell priming and reduced antitumor response. CD8α+ DC and pDC are needed for Ad5-TRAIL/CpG therapeutic efficacy (23), prompting us to test the responsiveness of these DC populations from DIO and ob/ob mice to CpG stimulation. We noted increased CD86, CD80, and CD40 expression on the surface of both CD8α+ DC and pDC from spleens of non–tumor-bearing lean and ob/+ mice after CpG stimulation (Fig. 3A and data not shown) as well as DC from ob/ob mice. In contrast, costimulatory molecule expression on either CD8α+ DC or pDC from DIO mice after CpG stimulation was not significantly different from that seen on unstimulated cells.
CD8+ T cells are also required for the reduced tumor burden after Ad5-TRAIL/CpG administration (16). Using an intravascular staining technique (18) to distinguish tissue-localized CD8+ T cells from those in the vasculature, we noted a significant increase in the frequency and number of CD8+ T cells in the tissue of tumor-bearing kidneys of lean mice after Ad5-TRAIL/CpG injection compared with PBS injection (Fig. 3B). This influx was not observed in DIO mice, but ob/ob mice also had increased CD8+ T cell infiltration after Ad5-TRAIL/CpG therapy, like the littermate controls. Nearly all of the CD8+ T cells detected in the kidney tissue had also upregulated CD11a (Supplemental Fig. 2), indicating they were “Ag experienced” (24) and infiltrating the tumor-bearing kidney in an Ag-specific manner. Collectively, these data suggest DIO mice have functionally impaired DC, correlating with decreased tumor infiltration by CD8+ T cells and the reduction in therapeutic efficacy.
Leptin neutralization in DIO mice restores therapeutic efficacy
The two models of obesity used in this study have yielded different responses to immunotherapy, and one obvious difference between the two models is the amount of endogenous leptin. DIO mice (and obese humans) have elevated levels of leptin compared with lean controls, whereas ob/ob mice lack leptin entirely (see Fig. 1C). To examine the extent to which leptin was responsible for the disparate results, DIO mice were given a soluble recombinant leptin receptor:Fc fusion protein (ObR:Fc) to reduce the amount of systemic leptin to the levels detected in lean mice (Fig. 4A). Tumor-bearing DIO mice were given ObR:Fc prior to Ad5-TRAIL/CpG or anti–CTLA-4 mAb therapy. Whereas DIO mice failed to show a response to either therapy, ObR:Fc-treated DIO mice now responded to both Ad5-TRAIL/CpG and anti–CTLA-4 mAb therapy, similar to normal weight controls (Fig. 4B, 4C). Further investigation into the restoration of therapeutic responsiveness after ObR:Fc administration found increased costimulatory molecule expression on CD8α+ DC and pDC after CpG stimulation (Fig. 4D, 4E) and increased CD8+ T cell infiltration of tumor-bearing kidneys after Ad5-TRAIL/CpG injection (Fig. 4F). Collectively, these data suggest leptin is one factor driving the lack of immunotherapy efficacy in DIO mice and (in part) explains why ob/ob mice are able to mount an antitumor response despite their profound obesity.
Currently, over one-third of the adult population in the United States is obese (5). The increased cancer risk associated with obesity and the growing use of cancer immunotherapies make it crucial to understand how obesity can impact antitumor immune responses. We have demonstrated that DIO in mice compromises the immune response to tumor immunotherapy. DIO mice have fewer functional DC, reduced tumor infiltration by CD8+ T cells, and ultimately an impaired response to therapy. Our data suggest that the chronically elevated levels of leptin observed in obesity have a detrimental effect on immunotherapy. Leptin is a 16-kDa protein hormone secreted by adipocytes that signals through its receptor, ObR, in the hypothalamus to signal satiety (25, 26). The absence of leptin, as in ob/ob mice, results in lack of satiation, continuous feeding, and ultimately obesity. However, with increasing adiposity, excess leptin can be measured in the circulation, resulting in leptin resistance and overeating (27). Most studies evaluating the role of leptin on immunity have focused on ob/ob mice, in which leptin is completely absent, or short-term, high-dose administration of leptin. Our use of DIO mice better recapitulates the chronic overexpression of leptin experienced during human obesity. Leptin is now recognized to have additional roles in multiple systems, including the immune system, as ObR is expressed on all innate and adaptive immune cells (28). Moreover, ObR is expressed on a wide variety of mouse and human tumors, and leptin can have a direct effect on tumor cells, promoting a variety of protumor consequences, including cytokine signaling, growth, and invasion (29–31). Understanding the effect of leptin on tumor and immune cells will identify intervention points to enhance immunotherapy efficacy in cancer patients. Further, leptin neutralization using mAb or soluble ObR constructs could serve as a means of increasing the therapeutic window in obese cancer patients.
Most preclinical testing of immunotherapies occurs in healthy mice lacking many of the immune modulating comorbidities present in humans, which may be one reason for the limited clinical success of many promising therapies developed and testing in preclinical animal models. Our data establish the need for addressing complications of obesity in the design of cancer therapies, and a number of reports have examined the impact of obesity on immunological responses during cancer or infection. Obesity causes a number of changes within the immune system that can reduce the generation and potency of systemic immunity, which warrant further investigation and consideration as a mechanism to reduced responsiveness to immunotherapy.
We thank the members of the University of Minnesota Center for Immunology for their helpful comments.
This work was supported by National Institutes of Health Grants CA109446 (to T.S.G.), CA009138 (to F.V.S.), and CA173657 (to A.W.) and the Climb 4 Kidney Cancer organization.
The online version of this article contains supplemental material.
Abbreviations used in this article:
renal cell carcinoma.
The authors have no financial conflicts of interest.