In this study, we examined a series of animal models with the goal of simulating a common, important, but understudied clinical scenario: systemic cancer recurrences after surgery. Using our optimal models of local and systemic recurrences, we evaluated the efficacy of an adjuvant systemic TGF-β inhibitor (1D11) in eliminating postoperative recurrences.
Each model displayed distinct advantages and disadvantages (summarized in ). Although previous approaches are reproducible and feasible, they fail to account for several important themes which render them of limited utility.
First, as previously described, metastatic focii are established during primary tumor growth [3
]. Unfortunately, multiple flank tumor models previous models poorly recapitulate these important steps in disease progression. For example, simultaneous injection of two flank nodules, a technique used widely in the literature, poorly mimics the time course of systemic metastasis developing, rather, it more closely resembles the circumstances of synchronous primary tumors (given that lesions are injected simultaneously). Similarly, the “rechallenge” approach fails to consider the biology of metastatic foci which exist prior to surgical resection.
A second consideration that is overlooked in traditional models is concomitant immunity. The concept of "concomitant immunity" was first reported by North and colleagues in 1984, and describes the acquired ability of a host with a progressive tumor to reject a second inoculum of the same tumor at a distant site [27
]. The mechanism of this immunity against tumors is primarily due to a protective T-cell memory that develops during the initial tumor exposure [28
]. Although this immune response is not powerful enough to eliminate the established tumor, it is able to eliminate a second inoculum. This immunity variably disappears (depending on the tumor and mouse strain), due to induction of suppressor cells, most notably T-regulatory cells [29
]. Our experiments support the early work described by both North and Bursuker and the more recent work conducted by Turk and her colleagues [27, Bursuker, 1986 #33, Turk, 2004 #45]. Due to concomitant immunity, we found it was difficult to inject multiple flank sites with cancer cells and have a reproducible model of metastasis. Further such findings likely confound data acquired using such approaches.
A third consideration involving previous models of postoperative cancer recurrences is perioperative immunosuppression. In our experiments, resecting the primary tumor indeed did provide a temporary window in which tumor growth occurred. Many studies suggest that surgery generates a transient immunosuppression that allows increased tumor growth [4
]. This window is thought to result from inflammatory, neural, and hormonal factors. One arm stems from general anesthesia, which has a role in decreasing natural killer cell activity [33
]. The other arm, surgery itself, impairs production of IL-2 [34
] and generates immune suppressor cells [35
]. These immunologic forces confound the already complicated task of developing clinically relevant animal models to study post-surgery recurrences.
To summarize, the balance between these complex factors results in "windows of opportunity" for the injected tumor cells to grow (), which are somewhat cell type dependent.
Figure 5 Representation of immunologic forces that influence animal models involving tumor resection. During a short period following initial tumor cell inoculation, a second inoculation will result in consistent growth. Following this period, growth of a second (more ...)
Based on our experiences and the considerations previously described, we still believe that an optimal model for post-operative systemic recurrences includes spontaneously metastatic flank tumors and surgical resection of the primary (flank) tumor focus. Accurate results can be obtained with diligently planned experiments and data collection techniques. This model is technically simple and parallels the sequence of events observed in human metastatic cancers. In several cell lines (LLC and LRK), we have characterized the metastatic (pulmonary) involvement which is observed following flank tumor resection. The most challenging aspect to this model pertains to monitoring systemic disease because metastatic lung lesions do not produce symptoms until the tumor burden is far advanced. This makes accurate monitoring of recurrent lung disease difficult, requiring periodic imaging (i.e. micro-CT scans) or timed sampling of experimental groups with necropsy to examine tumor infiltrating into the lung. In addition, we found that experiments which require sacrificing animals to measure the quantity of disease in the lung may necessitate 15 to 20 animals per treatment group to obtain statistically significant results. These issues add to the time needed and expense of the model. Despite hurdles, spontaneously metastatic models are predictable and resemble human cancer biology, making them potentially highly useful for studying systemic post-operative recurrence biology ().
Although encouraging, it is essential to understand our findings in the context of recent advances in cancer biology. Spontaneously arising orthotopic tumors in transgenic models have proven effective for studying tumor microenvironment, growth kinetics, and immune system changes. However, with the exception of breast and skin cancers, surgical resection of orthotopic lesions cannot be reliably or safely performed. Furthermore, these models often require prolonged periods for tumor development, which results in low throughput systems.
In order to validate our approach of studying systemic cancer recurrences, we investigated that the role of systemic TGF-β inhibition in preventing systemic recurrences. TGF-β is a cytokine with multiple immunological effects including powerful immunosuppressive features [36
]. It directly suppresses activation and maturation of innate and adaptive immune cells including CD3+ T cells, natural killer cells and antigen presenting cells [37
]. Tumors and suppressor myeloid cells have been implicated in the production of TGF-β [38
], and TGF-β inhibition has been proposed in patients as an adjuvant therapy to other chemotherapeutic and immunological approaches [39
]. This cytokine therapy has been thought to be safe, non-toxic and can be administered for long periods of time without side effects [40
We have over a decade long experience with TGF-β therapy and recently started a clinical trial in TGF-β inhibition of malignant mesothelioma. Therefore, we chose this agent because we were familiar with this approach, and it would inform a clinical trial combining cytoreductive surgery for malignant pleural mesothelioma with immunotherapy. Interestingly, in non-surgical models we found no observable decreases in systemic disease associated with TGF-β inhibition. However, when coupling TGF-β blockade with surgical resection we appreciated dramatic decreases in systemic tumor burden. Potential benefits of this approach may have been overlooked if not evaluated in a proper surgical model aimed at studying postoperative recurrences.
Incorporation of accurate systemic recurrence models following cancer surgery has been a challenge primarily due to the lack of systematically evaluated models. After assessing both current and newly proposed techniques, we conclude that the optimal model of systemic recurrence incorporates spontaneously metastatic cell lines followed by complete flank (primary) tumor excision. Once learned, these techniques offer realistic pre-clinical vehicles to study adjuvant therapies.