Local cancer recurrences following surgical resection of solid organ tumors affect nearly 250,000 patients each year.2
This cohort of patients does exceptionally poorly with dismal long-term outcomes.2
Despite enormous ramifications, there is a paucity of studies that examine therapies in the setting of post-surgical recurrences. In this study, we optimized a model of local recurrence (the “positive margin” model), and then used this model to evaluate the effect of two clinically relevant immunotherapies that influence the tumor microenvironment: systemic TGFβ blockade and COX-2 inhibition. The surgical setting had important effects on both approaches.
In the past, our group and others have studied postoperative local recurrences by incorporating “complete” flank tumor excision followed by monitoring for recurrent growth.7,14,23
We have found that with good technique, flank tumors of most tumor cell lines can be fully excised, and locally recurrent disease develops idiosyncratically over several weeks. Clearly, such models confound therapeutic trials and leave data acquisition inefficient.
Key characteristics of an ideal local recurrence surgical model include: (1) the ability to perform surgery on primary tumors without undue stress to the animal, (2) a high degree of technical feasibility (allowing high-throughput), (3) cost effectiveness, (4) reproducibility and (5) most important, similarity to humans cancer recurrence. The optimal animal model should also utilize an immunocompetent host given the complex inflammatory and immune responses that occur during the perioperative period such as the presence of an endogenous anti-tumor immune response (concomitant immunity),24
perioperative immune suppression,25
and inflammatory consequences associated with wounds and wound healing.10
These requirements make it difficult to use cancer models generated by recent advances in tumor modeling. Transgenic mice that develop orthotopic tumors have proven effective for studying oncologic concepts including carcinogenesis, tumor microenvironment, growth kinetics, and immune system changes. However, with the exception of breast and skin cancers, orthotopic tumors (for example lung, pancreas, liver, brain, colon, or the prostate tumors) are not conducive to surgical resection in mice. Additionally, these models often require prolonged periods for tumor development, resulting in low-throughput models that are ineffective for preclinical evaluations. Another advance in animal cancer modeling has been transfection or genetic manipulation of tumor models with marker genes (i.e., luciferase and green fluorescent protein) which may allow for monitoring recurrent lesions by imaging.26,27
Unfortunately, introduction of foreign proteins tends to increase the immunogenicity of the model, thus altering (in some circumstances completely inhibiting) tumor growth in wild type mice with intact immune systems. As a consequence, the vast majority of these imaging studies are performed with transfected cells xenografted into immunodeficient mice.27
Given these hurdles, we sought to develop a model of local cancer recurrence that would reproduce some (but not all) of the factors seen in human surgery. It is important consider that such tumor models deviate from human disease with respect to their tumor locations, variable genetic make-ups, and aggressive growth kinetics; however, cancer models in flank tumor locations allow for humane surgical resection and subsequent post-operative tumor observation. As demonstrated in our data, we began by performing an R0 resection (complete tumor resection), but were able to achieve local control easily, with cure rates approaching nearly 80% time. Although this technique best recapitulates what occurs in the human scenario, it was unfortunately unreliable in generating locally recurrent disease. Given this limitation, we deviated from the traditional human approach of “complete resection” and implemented a partial resection approach, by excising 95% of the tumor while leaving behind a small positive margin. This model is very similar to the clinical “debulking” scenario that might be used in ovarian cancer, brain cancer, and mesothelioma (an R2 resection), but also approximate the positive margin scenario occurring in human surgery. Although these drawbacks are present, this model likely does provide a more stringent model for preclinical adjuvant therapy evaluation given the relatively large amount of residual tumor.
Understanding these limitations, we still believe this approach was quite valuable. This resection model was technically feasible, while also being superiorly reproducible. Further all lab personnel were able to master the technique within several attempts. In total, the procedure required less than 10 min per mouse, and the growth curves were consistent among surgeons independent of surgical experience. This approach allowed for further analysis of postoperative tumor growth kinetics, time to disease progression and measurements of the postoperative tumor bed microenvironment. Most importantly, we were able to demonstrate important consequences of manipulating the postoperative tumor microenvironment by systemic inhibition of TGFβ or COX-2.
In addition to feasibility, this approach produces a biologically relevant model of local cancer recurrences and a surgically induced tumor microenvironment. With this consideration in mind, we sought to modulate two inflammatory forces commonly found in the recurrent tumor microenvironment that promote cancer recurrences. The first, TGFβ, is a cytokine with multiple known pro- and anti-tumor immunological effects.28,29
We have over a decade of experience with both anti-TGFβ therapy14,16,30,31
and COX-2 inhibition22,32,33
in mouse models. Furthermore, our group has recently begun a clinical trial using an anti-TGFβ monoclonal antibody GC-1008 (Genzyme) in patients with malignant mesothelioma and a trial combining COX-2 inhibition with type I interferon immune-gene therapy approaches. Unfortunately, like many early phase trials, drug evaluation has been implemented in patients with advanced, refractory disease. However, results of previous studies suggest that maximum immunotherapy efficacy is obtained when administered in the setting of minimal disease.34
To this extent we have become interested in pursuing immunotherapy following surgery, which may create “minimal disease” state.
Given that platelets account for a major deposition of TGFβ into the wound at the time of surgery,35
we predicted that beginning TGFβ blockade before
would provide maximum benefits. As hypothesized, when 1D11 was administered before
surgery, we observed much more dramatic inhibition of growth than when the antibody was begun after
surgery. However, our model allowed us to observe a major complication associated with preoperative dosing—dramatic wound breakdown. These findings are consistent with the known role for TGFβ in wound healing,35,36
but were missed in our previous studies where TGFβ was not blocked in the presence of an active wound.14
The use of the positive margin model was instrumental in confirming the theoretical hypothesis involving TGFβ inhibition’s impact on wounding, and will help in developing protocols that maximize benefits while minimizing complications.
The second adjuvant interrogated was a systemic inhibitor of cyclooxygenase-2 (COX-2). Cyclooxygenases catalyzes the rate-limiting step in the formation of prostaglandins which have known immunosuppressive roles.37,38
Two forms exist: the constitutive COX-1 and the inducible COX-2. Compared with COX-1, COX-2 leads to increased formation of PGE2 from arachidonic acid which appears to contribute to the cancer phenotype.37,38
COX-2 inhibition presented a second therapeutic dilemma. Whereas we and others have clearly shown that blockade of COX-2 can inhibit primary tumor growth,22,33
COX-2 is strongly upregulated in the wound microenvironment after surgery where it presumably plays a positive role in wound healing and tumor recurrence.3
Using our local recurrence model, we were able to experimentally test the effect of perioperative administration of the COX-2 inhibitor celecoxib. Like TGFβ blockade, perioperative celecoxib was effective in slowing tumor recurrence. However, in this case, no adverse wound healing effects were observed, suggesting that this approach has potential merit for testing in clinical trials involving surgery. When we treated recurrent tumors postoperatively, the efficacy of COX-2 inhibition was essentially lost. This highlights a particular issue as most surgeons are reluctant to utilize a COX-2 inhibitor perioperatively due bleeding concerns.
In summary, adjuvant therapies aimed at limiting postoperative local recurrences have unfortunately been inadequately evaluated in the past, primarily due to suboptimal models. Instead of testing adjuvant therapies in preclinical models that incorporate at least some of the complex effects of surgery and the positive margin microenvironment, data has been extrapolated from non-surgical models. It is possible that the lack of appropriate surgical models can explain the marginal efficacy of many adjuvant therapies.39
This report demonstrates that the positive margin model is an efficient and biologically relevant preclinical tool useful for studying systemic and local therapies addressing post-operative cancer recurrences. We appreciated strong adjuvant effects of both COX-2 inhibitors and TGFβ blockade. More importantly, however, this model provided important information involving wounding issues and adjuvant timing which would have been overlooked in previous cancer models.