In the present study, we characterized the ability of SR/CR mice to resist additional lethal cancer cell lines in vivo, and showed that SR/CR mice are able to resist these cell lines at higher doses than WT mice. However, SR/CR mice were able to resist some cancer cell lines at exceptionally high doses (high-MTD), whereas other cancer cell lines were only resisted at moderate or lower doses (low-MTD). We found that co-injection with either S180 or CFAF, both capable of inducing massive leukocyte infiltration specifically in SR/CR mice, was able to increase the level of resistance of SR/CR mice to cell lines with otherwise low-MTD.
Cancer cells may undergo selection to avoid detection by the immune system, a process termed cancer immuno-editing that may drive the progression of malignancy [10]. A variety of tumor-derived factors may contribute to immunosuppressive processes that may extend immune evasion from the primary site to peripheral sites in patients with cancer [11]. The results from our experiments suggest that cancers with low-MTD escape from SR/CR anticancer immunity because they do not produce sufficient chemoattractants. These low-MTD cancer cell lines are capable of being eradicated in SR/CR mice at lower doses, presumably by the less than 2 × 10e6 resident leukocytes usually present locally in the peritoneal cavity. At higher doses, however, the growth of these low-MTD cancer cell lines may outpace the killing ability of these limited resident leukocytes. This is in contrast to cell lines such as S180 that are able to induce a very large infiltration of leukocytes. The larger numbers of infiltrating SR/CR leukocytes are apparently capable of killing a much larger number of cancer cells.
Although the outcome of the co-injection experiment with S180 and CFAF was somewhat unexpected, the results, coupled with our previous work demonstrating that S180, MethA, and LL/2 were killed in an in vitro assay [2], suggest that the ability to induce leukocyte infiltration may be the most significant factor between these cancer cells in their ability to be resisted by SR/CR mice. There appear to be common surface properties that allow a variety of cancer cells to be recognized, bound, and destroyed by SR/CR leukocytes when they are in close proximity (Figure 6). Our results demonstrate that S180 only enhances resistance against LL/2 locally in SR/CR mice and argues that the ability of S180 to induce leukocyte infiltration is the critical event in augmenting resistance. While immune infiltration is clearly important for the eradication of cell lines such as LL/2 and MethA, we cannot completely exclude the possibility that co-injection with S180 or CFAF also activates the effector mechanism of SR/CR leukocytes, in addition to the induction of their infiltration.
The results also suggest that there is a diffusible chemoattractant gradient established by some cancer cells. CFAF from S180 contains high levels of these chemoattractants that are specific for SR/CR leukocytes. Our results indicate that the most active chemoattractants are molecules smaller than 5 kD that are heat sensitive. It is currently unclear if these diffusible chemoattractants were produced by active cellular secretion [11, 12], by passive "surface shedding", a physical process, from cancer cells [13–16] or indirectly by the interaction between cancer cells and stromal tissues. Nevertheless, CFAF offers a good platform for further biochemical purification and identification of these chemoattractants. Apparently, chemoattraction of SR/CR leukocytes is a separate process from recognition of the common cancer cell surface properties that allow for local binding and eradication of SR/CR leukocytes, since some cancer cells can lose the former process while retaining the properties of the latter. This ability to induce leukocyte infiltration through chemoattraction appears to be the reason that SR/CR leukocytes are effective as a systemic therapy against established S180 cancers, but are only locally effective against cancers like LL/2.
The cancer/immune cell interaction involves events on either side that influence the ability of the immune system to eradicate the cancer. On the cancer side, malignant lesions may range from being highly immune-attractive (S180) to inducing little immune infiltration (LL/2). Cancer cells may develop mechanisms that prevent migration of leukocytes to the site of the cancer, either by turning off the production of inflammatory molecules that can act as chemoattractants or by producing molecules that actively inhibit immune cells, such as TGF-β [17]. Our results indicate that cancer cells and CFAF may also produce molecules that are directly inhibitory to WT leukocytes (Figure 4C). On the host side, leukocytes themselves can vary significantly in any of the three required stages of leukocyte response. If leukocytes have a defect in infiltration to the cancer site, recognition of the cancer, or deployment of their effector mechanisms an effective anticancer response to protect the host cannot take place. For example, leukocytes may infiltrate the cancer site but may be unable to recognize and kill the cancer cells. This has been reported in some melanoma patients, in whom despite having melanoma-specific T cells infiltrating the tumor lesions, tumor rejection rarely occurs [18]. Additionally, the responsiveness of host leukocytes may be influenced by genetics, aging and environmental factors.
Our SR/CR model system is very interesting in light of many recent reports that there is a positive correlation between tumor infiltrating lymphocytes and the survival of patients with melanoma, ovarian cancer, bladder cancer, glioma, and colon cancer [19–23]. It is worth noting that in these examples of human cancers the correlation is with cells of the adaptive immune system, specifically T lymphocytes, while in the SR/CR mouse the resistance mechanism is mediated by the innate immune system. A recent study performed by Galon et al. is particularly intriguing as it specifically links activation of the cellular immune response, including macrophages of the innate immune system, to patient outcomes in colorectal cancer [24]. They find a significant correlation between expression of genes of the Th1 response and a beneficial outcome in risk of relapse after complete removal of the tumor.
The best case scenario for an anticancer protection mechanism in a host would be having cancer cells that secrete a chemoattractant and having leukocytes that can infiltrate, recognize the cancer cells as foreign, and completely destroy the cancer. However, the absence of one or more of these factors could lead to an unfavorable host/cancer interaction enabling the cancer to escape immunosurveillance resulting in progression of the disease. When there is no recognition of cancer cells by leukocytes, manipulation of other processes, such as infiltration, will not improve host survival. However, if infiltration is the only deficiency, as we observe in SR/CR mice challenged with LL/2, local delivery of host leukocytes or establishment of a chemoattractant gradient at the cancer site could achieve therapeutic benefit.