TLR Agonist Therapy of Metastatic Breast Cancer in Mice : Journal of Immunotherapy

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Basic Studies

TLR Agonist Therapy of Metastatic Breast Cancer in Mice

Klinman, Dennis M.; Goguet, Emilie; Tross, Debra

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Journal of Immunotherapy 46(5):p 170-177, June 2023. | DOI: 10.1097/CJI.0000000000000467
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Single Toll-like receptor (TLR) agonists are being used to treat human cancer. This work establishes that a combination of TLR7/8 plus TLR9 agonists, when combined with other antitumor modalities, significantly improves outcomes in mice with advanced metastatic breast cancer. These preclinical findings strongly support clinical studies of TLR agonist combinations in patients with advanced disease.


Breast cancer afflicts nearly 1 in 8 American women and is the cause of over 40,000 deaths/year.1 Treatment of early localized disease includes surgery, radiation, hormones and/or chemotherapy. These approaches are highly successful in the treatment of primary tumors, but outcomes are less favorable once the tumor has metastasized to the lungs, bones, liver, or brain.1–4

Animal models provide invaluable tools for assessing the therapeutic potential of novel anti-cancer strategies. Both human and murine tumors become increasingly resistant to therapy as they grow, metastasize, and develop immunosuppressive microenvironments that interfere with the tumoricidal activity of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Thus, treatments that successfully control the growth of small murine tumors (<100 mm3) fail to alter disease progression in animals or patients with metastatic disease.1,5–7 Outcomes better predictive of human efficacy are obtained by using orthotopic implantation models and in which therapy is delayed until the primary tumor has become large (>250 mm3) and metastasized to distant sites.5,8

66cl4 is a derivative of the highly aggressive 4T1 breast cancer cell line. Orthotopic implantation of these cells into the mammary gland of syngeneic BALB/c mice induces a disease similar to human breast cancer.6,9 The tumor grows for several weeks before becoming locally invasive and metastasizing to the lungs. The response of 66cl4 tumors to therapy at this stage of disease correlates with human clinical outcomes.6,9 Previous studies indicate that neither 66cl4 nor metastatic human breast cancer respond well to immunotherapy.6,9–11 Indeed, many therapies that successfully eradicate other murine tumors have little effect on 4T1-derived cells. This reflects, in part, the expression of PD-L1 by 66cl4 cells (a trait shared by one-third of human breast cancers), as PD-L1 down-modulates tumoricidal CTLs and NK cells.12,13

Our lab previously documented that intratumoral injection of a combination of TLR7/8 and TLR9 agonists was effective in the treatment of primary CT26 colon carcinoma and B16F10 melanoma.14 TLR agonists increase the number of tumoricidal NK and CD8 T cells infiltrating the tumor bed while reducing the number and activity of immunosuppressive myeloid-derived suppressor cells (MDSCs) and Tregs.15–19 The current work was undertaken to examine the effect of this therapy on the host’s response to metastatic 66cl4 tumors. Metastatic lung tumors are critically important therapeutic targets since (i) the lungs are the most common site of human cancer metastases and (ii) treatment can be delivered directly through inhalation.1,6 Current results demonstrate that tumor burden is significantly reduced when these agonists are delivered to both the primary tumor and sites of metastases, an effect that is enhanced by the co-delivery of cyclophosphamide and anti-PD-L1.



3M-052, 3M-058, and anti-PD-L1 were supplied by 3M Drug Delivery Systems Division. Endotoxin-free phosphorothioate ODN was synthesized at the Core Facility of the Center for Biologics Evaluation and Research, Food and Drug Administration (Silver Spring, MD). The sequence used was: CpG ODN 1555 (5′-GCTAGACGTTAGCGT-3′).

Mice and Tumor Cell Lines

66cl4 cells were used with permission from Dr Fred Miller of the Karmanos Center of Wayne State University. A luciferase-tagged construct of these cells was provided by Dr Shoukat Dedhar of the British Columbia Cancer Agency. A 10 to 12–week-old BALB/c mice, purchased from the Jackson Laboratory and bred in-house, were used in all experiments involving 66cl4-luc cells. Cells were maintained in DMEM media supplemented with 10% FCS, 0.3 mg/ml L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 10 mM Hepes, and 10–5 M 2-mercaptoethanol and cleared for use in animals by the animal health diagnostic laboratory of Frederick National Lab. All studies were approved by the National Cancer Institute Frederick Animal Care and Use Committee.

66cl4- Tumor Experiments

106 66cl4-luc cells were implanted into the fourth mammary fat pad of 10 to 12-week-old female BALB/c mice. Tumor growth was monitored weekly by bioluminescent imaging. Animals were evaluated individually to avoid photon contamination between subjects 11 minutes after IP injection of 100 mg/kg of D-luciferin (conditions found to be optimal in preliminary experiments). A shield was placed over the abdomen to evaluate lung metastases, as this prevented photon contamination from the primary tumor. In some experiments, tumors were surgically removed when the primary tumor reached 300 mm3 (volume is calculated using the following formula: length-mm× width-mm×height-mm). In other experiments, treatment was initiated when tumor size exceeded 250 mm3, and bioluminescent imaging showed a signal >2.0×105 photons/s, which was universally associated with the presence of lung metastases based on histologic analysis (data not shown). Primary tumors were treated twice per week with a mixture of 100 µg 3M-052 plus 200 µg CpG ODN in 50 µl of PBS delivered intratumorally (IT) through a 27G needle. Some mice received a single IP injection of cyclophosphamide (100 mg/kg), while other mice received anti-PD-L1 (10 mg/kg, IP) twice weekly (Fig. 1). Mice were anesthetized and intubated with a soft 20G catheter to administer 25 µg of 3M-058 and 100 µg of CpG ODN to the lungs. Mice were carefully monitored for adverse side effects. Pulmonary therapy was repeated weekly if lung bioluminescence remained >2.0×105 P/s and mice were within 12% of their pretreatment body weight.

Bioluminescent monitoring of tumor progression. 106 66cl4-luc tumor cells were surgically implanted into the fourth mammary fat pad of BALB/c mice. Tumor growth was monitored weekly by bioluminescence. To quantify pulmonary metastases, a shield was placed over the abdomen to prevent photon contamination originating from the primary tumor. Data show changes in photon intensity of the primary tumor (top row) and lung metastases (bottom row) in an animal whose primary tumor was surgically removed 23 days postimplantation. Note that a signal greater than 2.0×105 photons/s correlated with the presence of lung metastases as determined by histopathology upon necropsy.

Statistical Analysis

P values for each experimental group were determined by comparison with the PBS control group using an unpaired student’s t test. Survival was analyzed using the Log-rank test with the Bonferroni correction.


Growth of Orthotopic 66cl4 Tumors

To examine the progression of both primary and metastatic tumors,106 luciferase-expressing 66cl4 carcinoma cells were implanted into the fourth mammary fat pad of syngeneic BALB/c mice. Tumor growth was monitored weekly by bioluminescent imaging (Fig. 1).

Untreated 66cl4 tumors grew to terminal size (>2 cm as defined by the NCI Animal Care and Use Committee) in ~7 weeks.20 Consistent with previous reports, these tumors seeded lung metastases 17 to 20 days postimplantation.6 If the primary tumor was resected before day 17, lung metastases did not develop, and surgery was curative (similar to localized human breast tumors, data not shown). If the primary tumor was not removed, all mice developed extensive lung metastases (verified histologically postmortem). Metastatic lesions accumulated over time but generally remained smaller than the primary tumor (Fig. 1).

Effect of TLRcomb in Mice With Advanced 66cl4 Tumors

Our group and others previously showed that intratumoral injection of TLR7/8 and/or TLR9 agonists substantially reduced the proliferation of large (>250 mm3) CT26 carcinomas.14–19 The 66cl4 murine model of human breast cancer enabled the study of animals with highly invasive primary and metastatic disease. Initial experiments compared the benefits of intratumoral injection with CpG ODN plus 3M-052 (hereafter TLRcomb) to surgical resection of the primary tumor. Treatment was initiated in animals based on the following 2 criteria: (i) their tumors were a minimum of 250 mm3 in volume and (ii) bioluminescent imaging established that pulmonary metastases were present (Fig. 2).

General experimental protocol. 106 66cl4-luc tumor cells were surgically implanted as described in Figure 1. (A) Surgery or treatment with TLRcomb was initiated when the primary tumor exceeded 250 mm3 in size (dotted arrow), and lung metastases were confirmed by bioluminescence. (B) Surgery or treatment with TLRcomb was initiated when the primary tumor exceeded 300 mm3 (dotted arrow). Lung metastases (confirmed by bioluminescence) were treated with TLRcomb every 10 days. If tumor progression was observed, additional forms of antitumor therapy were substituted, as shown.

As seen in Figure 3, both primary and metastatic lesions progressed in untreated mice. Mice injected with PBS alone succumbed to cancer ~3 weeks after therapy was initiated. Animals treated bi-weekly with intratumoral TLRcomb survived significantly longer than untreated controls (P<0.01, Fig. 3C). TLRcomb treatment reduced tumor burden both locally and in the lungs, the latter presumably due to decreased seeding from the primary lesion and/or systemic improvement in T cell immunity. While tumor resection was curative in mice before day 17, that was not the case once cancer had metastasized. Despite the >100-fold decrease in bioluminescent signal intensity of the primary tumor achieved by surgery, locally invasive cancer cells regrew (or were re-seeded from pulmonary metastases). While significantly prolonging survival, surgery proved to be less effective at controlling disease progression than TLRcomb therapy (Fig. 3).

Effect of treating large primary tumors with TLRcomb. 106 66cl4-luc tumor cells were surgically implanted on day 0, as described in Figure 1. Mice were monitored daily, and their tumors were either surgically resected (N=5, solid triangle) or injected IT with TLRcomb (N=4, open circle) ~3 weeks later when they exceeded 250 mm3 in size and the presence of lung metastases confirmed by bioluminescence (solid arrow). (A) Changes in tumor mass over time were evaluated by bioluminescence in comparison to untreated tumor-bearing animals (N=7, open triangle). Mice were removed from the study and euthanized once their tumors exceeded 2 cm in length. (B) The progression of metastatic lung tumors was evaluated by bioluminescence. (C) The survival of the mice described above was analyzed by a Bonferroni corrected Log-rank test. *P<0.05, **P<0.01 vs. untreated tumor bearing controls, +; P<0.05 vs. tumor resected mice.

To verify that the combination of CpG ODN plus 3M-052 provided optimal control of 66cl4 tumors, the effect of each component was compared with the combination. While individual TLR agonists prolonged survival and reduced tumor burden (Fig. 4A, C), the combination was significantly more effective than either component alone (Fig. 4). Three weeks after the initiation of the treatment (the last timepoint when all untreated controls were still alive), TLRcomb reduced pulmonary tumor load by 19-fold vs. a 2–5-fold reduction by 3M-052 or CpG ODN (Fig. 4B).

Effect of treating large primary tumors with TLR agonists. 106 66cl4-luc tumor cells were surgically implanted as described in Figure 1. Mice were monitored daily, and their tumors were treated with CpG ODN (N=5 left-shaded circles), 3M-052 (N=5, right-shaded circles), or TLRcomb (N=4, solid circles) when they exceeded 250 mm3 in size and the presence of lung metastases confirmed by bioluminescence. (A) Change in the mass of the primary tumor over time, evaluated by bioluminescence. (B) Change in mass metastatic lung tumors over time, evaluated by bioluminescence. (C) Survival. *P <.05, **P<0.01 vs. untreated tumor-bearing controls, +; P<0.05 vs. mice Rx’d with either TLR agonist alone.

Effect of TLRcomb on the Progression of Pulmonary Metastases

Over the course of these experiments, it was observed that even when the primary tumor regressed, the metastatic disease still progressed in the lungs. Two strategies were pursued to enable long-term study of lung metastases: (i) surgical resection of the primary tumor and (ii) bi-weekly intratumoral injection of TLRcomb. Debulking the primary tumor reduced its size by >100-fold, but lung metastases persisted (Figs. 3A, B, 5B). By comparison, delivering TLRcomb to the lungs after surgical resection of the primary tumor dramatically reduced pulmonary tumor burden (Fig. 5B). Whereas bi-weekly intratumoral injection of TLRcomb reduced the size of primary lesions by ~5-fold adding intrapulmonary TLRcomb resulted in a substantial reduction in both primary and metastatic disease (Fig. 5).

Effect of treating metastatic lesions with intrapulmonary TLRcomb. 106 66cl4-luc tumor cells were surgically implanted as described in Figure 1. Mice were monitored daily, and their tumors were either surgically resected (N=10, squares) or treated with TLRcomb (N=12, circles) when they exceeded 300 mm3 in size and the presence of lung metastases confirmed by bioluminescence. Half of the mice in each group were also treated weekly with intrapulmonary TLRcomb (solid symbols). (A) Change in the mass of the primary tumor over time, evaluated by bioluminescence. (B) Change in mass metastatic lung tumors over time, evaluated by bioluminescence.

Effect of Combining TLRcomb With Other Immunomodulators

Repeated TLRcomb treatment of the lungs caused toxicity in some animals characterized by weight loss, hunched posture, and rapid shallow breathing. Interestingly, this was not observed after intrapulmonary administration of TLRcomb to normal mice, suggesting that toxicity was caused by either a tumor-dependent reduction in pulmonary function or the inflammation induced by effective tumor therapy. Nevertheless, the treatment protocol was modified to reduce the frequency of intrapulmonary therapy and substitute the less toxic TLR7/8 agonist 3M-058 for 3M-052 (Fig. 2B). Bi-weekly intratumoral delivery of TLRcomb combined with intrapulmonary instillation of CpG plus 3M-058 (hereafter TLRcomb2) reduced the size of primary tumors by 5–10 fold (Fig. 6A) and delayed the growth of pulmonary metastases by several weeks (Fig. 6B and data not shown). Despite this success, treatment efficacy eventually waned, and mice succumbed to the disease.

Effect of additional immunotherapies on the progression of primary and metastatic lesions. 106 66cl4-luc tumor cells were surgically implanted as described in Figure 1. Mice were monitored daily, and their tumors were treated when they exceeded 300 mm3, and the presence of lung metastases was confirmed by bioluminescence. (A) Progression of primary tumors, evaluated by bioluminescence. (B) Progression of metastatic disease, evaluated by bioluminescence. (C) Survival curves. Survival was analyzed using the Log-rank test with the Bonferroni correction. **P<0.01, ***P<0.001 compared with No Rx control, ++; P<0.01 vs mice treated with TRLcomb alone. TLR Rx consisted of a combination of intratumoral TLRcomb plus intrapulmonary TLRcomb2, as described in Figure 2. Treatment groups: CY only (N=7, open square); anti-PD-L1 only (N=6, open diamond); TLRcomb (N=12, gray circle); TLRcomb + CY (N=4, solid square); TLRcomb + anti-PD-L1 (N=6, solid diamond); TLRcomb + CY + anti-PD-L1 (N=4, solid circle); No Rx (N=8, open triangle).

In an effort to further prolong survival, other immunomodulatory therapies were tested. These included administering a single 100 μg/gm dose of cyclophosphamide (CY) and/or bi-weekly injections of anti-PD-L1 to mice when TLR treatment was paused due to toxicity. Treating with CY or anti-PD-L1 alone had no significant impact on tumor growth. Adding CY or anti-PD-L1 to TLRcomb therapy increased survival by 1 to 2 weeks (Fig. 6C). Optimal tumor control and survival were achieved when TLR agonist therapy was supplemented by anti-PD-L1 together with CY (Fig. 6C).


This work evaluated the effect of TLR agonist therapy on primary and metastatic breast cancer in mice challenged with invasive 66cl4 tumors. Treatment was initiated only after the primary tumor exceeded 250 mm3 in size and had seeded metastases to the lungs (verified by bioluminescence and subsequent histologic analysis).1,5 While significantly prolonging the survival of mice with advanced disease is quite difficult, therapies found to be effective under these demanding conditions are more likely to prove successful in clinical trials.3,5,6,9

TLRcomb consists of 2 components: the TLR7/8 agonist 3M-052 plus the TLR 9 agonist CpG ODN. TLR7 is primarily expressed by plasmacytoid dendritic cells, TLR8 by monocytes, monocyte-derived (m)DCs, macrophages and Langerhans cells, and TLR9 by DCs, B cells, monocytes, and mast cells.21–24 Synthetic agonists designed to stimulate TLR7 typically trigger TLR8 as well and induce the release of IL-12 and TNFα by monocyte-derived dendritic cells and/or plasmacytoid dendritic cells.25,26 Many TLR7/8 agonists also enhance antigen-presenting cell (APC) expression of co-stimulatory molecules and DC migration, which in turn facilitates the induction of Th1 immune responses.27,28 Synthetic oligonucleotides that express CpG motifs trigger TLR9 and elicit a Th1-dominated immune response characterized by the production of proinflammatory cytokines (including IL-12, IFNα, and TNFα) and the upregulation of co-stimulatory (CD80 and CD86) and MHC class I and II molecules.29–31 By shifting the immune milieu from immunosuppressive to inflammatory, TLR agonists support tumor elimination.14,18

A growing body of evidence suggests that the antitumor activity of TLR agonists is optimized by (i) using them in combination and (ii) delivering them directly into the tumor bed18,32–37 In this context, our lab found that the combination of TLR7/8 and TLR 9 agonists synergistically enhanced the elimination of large primary CT26 and B16F10 tumors.38 Combination TLR therapy increased the number of tumoricidal NK and CD8 T cells infiltrating the tumor by 4-8 fold.18,39 Within the tumor bed, TLR agonists converted immunosuppressive MDSCs into inflammatory macrophages and reduced the frequency of Tregs,18,37,40 which would otherwise suppress the activity of cytotoxic CTLs and NK cells.14,41,42 MDSCs commonly infiltrate human and murine mammary tumors, constituting up to 5% of a total tumor mass.43 Strategies that deplete MDSCs have been shown to improve antitumor immunity.18,37,44

This work examined the effect of various TLR agonists on the growth of large primary 66cl4 tumors. Previous studies established that this form of therapy is tumor cell type agnostic: efficacy has also been documented against lung, colon, brain, and skin cancers.15–17 Current results show that intratumoral delivery of TLRcomb is more effective than either 3M-052 or CpG ODN alone at reducing tumor growth and prolonging survival (Fig. 4). The potential benefit of intratumoral TLR agonist therapy for breast cancer is being evaluated by the ongoing NCT03788083 clinical trial as are other forms of intratumoral monotherapy (NCT02531425, NCT03788083).45 While intratumoral TLRcomb therapy prolonged survival and slowed the growth of distant metastases, it was not curative (Figs. 4-5-6). In an effort to better control pulmonary metastases, the effect of delivering TLRcomb selectively to the lungs was examined (mimicking the effect of inhalational therapy in humans).40 Intrapulmonary treatment significantly slowed the progression of lung disease but had little effect on the primary tumor (reinforcing the importance of intratumoral TLR administration, Fig. 5 A, B).

To improve the survival of mice with advanced disease, TLRcomb was administered to both the lungs and the primary tumor. Targeting both sites reduced tumor burden and more than doubled lifespan (Fig. 4). Unfortunately, efforts to further improve outcomes by increasing the dose and/or frequency of TLRcomb administration were limited by toxicity. Weight loss provided the earliest indication of an adverse effect. If intrapulmonary therapy was continued, toxicity worsened, culminating in death. Postmortem analysis of mice with pulmonary toxicity found that tumor nodules in the lungs were necrotic and surrounded by extensive inflammatory infiltrates (data not shown). Toxicity could be avoided by pausing intrapulmonary TLRcomb therapy. Of interest, neither weight loss nor other adverse outcomes were observed when TLRcomb was repeatedly instilled into the lungs of non–tumor-bearing mice. The observation that intrapulmonary TLRcomb was toxic only in mice with metastatic tumors has several possible explanations. One is that the inflammation induced by TLR agonists reduced air exchange in mice whose pulmonary function was already compromised by the tumor. Another is that the tumor destruction mediated by TLRcomb therapy led to the release of DAMPs, cytokines, and/or tumor antigens that directly or indirectly promoted further inflammation. Efforts to differentiate among these alternatives are underway. In this context, interventional radiologists have enabled the intratumoral delivery of therapeutic agents to cancers in the breast, lungs, lymph nodes, and liver.46–48 Intratumoral administration is likely to lessen systemic exposure and thus reduce the toxicity observed in mice after intratracheal TLRcomb delivery. Should this strategy prove successful, it could be readily extended to the treatment of other sites of metastases.

Tumor growth in the lungs resumed when intrapulmonary TLRcomb was paused to avoid toxicity (data not shown). To prevent that outcome, additional therapy was instituted consisting of low-dose cyclophosphamide and/or bi-weekly anti-PD-L1. Low-dose CY mediates tumor suppression by down-regulating Tregs.10,49 Anti-PD-L1 reduces checkpoint inhibition of tumoricidal CTLs, thereby enhancing antitumor immunity.12,13,50 In this context, PD-L1 is expressed by one-third of human breast cancers and by 66cl4 tumor cells.1,11,44,51

Treating large established tumors with CY or anti-PD-L1 in the absence of TLRcomb had no significant impact on disease progression. When all 3 therapies were combined, the survival of mice with advanced disease improved by 4-fold and durable cures was achieved in a subset of animals. Previous efforts to treat advanced breast cancer with immunotherapy in the absence of cytotoxic agents have met with little success. Current findings demonstrate that TLRcomb can be used in combination with such therapies to significantly improve the survival of mice with locally invasive primary tumors and lung metastases. These findings strongly support further clinical testing of this regimen in patients with advanced disease.


The authors thank Dr John Vasilakos and his colleagues at 3M Drug Delivery Systems for kindly providing the TLR7 agonists used in this work. The assertions herein are the private ones of the authors and are not to be construed as official or as reflecting the views of the National Cancer Institute at large.

Conflicts of Interest/Financial Disclosures

None reported. All authors have declared there are no financial conflicts of interest with regard to this work.


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TLR agonists; metastatic cancer; mouse