Cancer resistance of SR/CR mice in the genetic knockout backgrounds of leukocyte effector mechanisms: determinations for functional requirements.

BACKGROUND: Spontaneous Regression/Complete Resistant (SR/CR) mice are a colony of cancer-resistant mice that can detect and rapidly destroy malignant cells with innate cellular immunity, predominately mediated by granulocytes. Our previous studies suggest that several effector mechanisms, such as perforin, granzymes, or complements, may be involved in the killing of cancer cells. However, none of these effector mechanisms is known as critical for granulocytes. Additionally, it is unclear which effector mechanisms are required for the cancer killing activity of specific leukocyte populations and the survival of SR/CR mice against the challenges of lethal cancer cells. We hypothesized that if any of these effector mechanisms was required for the resistance to cancer cells, its functional knockout in SR/CR mice should render them sensitive to cancer challenges. This was tested by cross breeding SR/CR mice into the individual genetic knockout backgrounds of perforin (Prf-/-), superoxide (Cybb-/), or inducible nitric oxide (Nos2-/). METHODS: SR/CR mice were bred into individual Prf-/-, Cybb-/-, or Nos2-/- genetic backgrounds and then challenged with sarcoma 180 (S180). Their overall survival was compared to controls. The cancer killing efficiency of purified populations of macrophages and neutrophils from these immunodeficient mice was also examined. RESULTS: When these genetically engineered mice were challenged with cancer cells, the knockout backgrounds of Prf-/-, Cybb-/-, or Nos2-/- did not completely abolish the SR/CR cancer resistant phenotype. However, the Nos2-/- background did appear to weaken the resistance. Incidentally, it was also observed that the male mice in these immunocompromised backgrounds tended to be less cancer-resistant than SR/CR controls. CONCLUSION: Despite the previously known roles of perforin, superoxide or nitric oxide in the effector mechanisms of innate immune responses, these effector mechanisms were not required for cancer-resistance in SR/CR mice. The resistance was functional when any one of these effector mechanisms was completely absent, except some noticeably reduced penetrance, but not abolishment, of the phenotype in the male background in comparison to female background. These results also indicate that some other effector mechanism(s) of granulocytes may be involved in the killing of cancer cells in SR/CR mice.

Impact of sex, MHC, and age of recipients on the therapeutic effect of transferred leukocytes from cancer-resistant SR/CR mice.

BACKGROUND: Spontaneous Regression/Complete Resistant (SR/CR) mice are resistant to cancer through a mechanism that is mediated entirely by leukocytes of innate immunity. Transfer of leukocytes from SR/CR mice can confer cancer resistance in wild-type (WT) recipients in both preventative and therapeutic settings. In the current studies, we investigated factors that may impact the efficacy and functionality of SR/CR donor leukocytes in recipients. RESULTS: In sex-mismatched transfers, functionality of female donor leukocytes was not affected in male recipients. In contrast, male donor leukocytes were greatly affected in the female recipients. In MHC-mismatches, recipients of different MHC backgrounds, or mice of different strains, showed a greater negative impact on donor leukocytes than sex-mismatches. The negative effects of sex-mismatch and MHC-mismatch on donor leukocytes were additive. Old donor leukocytes performed worse than young donor leukocytes in all settings including in young recipients. Young recipients were not able to revive the declining function of old donor leukocytes. However, the function of young donor leukocytes declined gradually in old recipients, suggesting that an aged environment may contain factors that are deleterious to cellular functions. The irradiation of donor leukocytes prior to transfers had a profound suppressive effect on donor leukocyte functions, possibly as a result of impaired transcription. The cryopreserving of donor leukocytes in liquid nitrogen had no apparent effect on donor leukocyte functions, except for a small loss of cell number after revival from freezing. CONCLUSION: Despite the functional suppression of donor leukocytes in sex- and MHC-mismatched recipients, as well as old recipients, there was a therapeutic time period during the initial few weeks during which donor leukocytes were functional before their eventual rejection or functional decline. The eventual rejection of donor leukocytes will likely prevent donor leukocyte engraftment which would help minimize the risk of transfusion-associated graft-versus-host disease. Therefore, using leukocytes from healthy donors with high anti-cancer activity may be a feasible therapeutic concept for treating malignant diseases.

Breast tumor cells isolated from in vitro resistance to trastuzumab remain sensitive to trastuzumab anti-tumor effects in vivo and to ADCC killing.

An understanding of model systems of trastuzumab (Herceptin) resistance is of great importance since the humanized monoclonal antibody is now used as first line therapy with paclitaxel in patients with metastatic Her2 overexpressing breast cancer, and the majority of their tumors has innate resistance or develops acquired resistance to the treatment. Previously, we selected trastuzumab-resistant clonal cell lines in vitro from trastuzumab-sensitive parental BT-474 cells and showed that cloned trastuzumab-resistant cell lines maintain similar levels of the extracellular Her2 receptor, bind trastuzumab as efficiently as the parental cells, but continue to grow in the presence of trastuzumab and display cell cycle profiles and growth rates comparable to parental cells grown in the absence of trastuzumab (Kute et al. in Cytometry A 57:86-93, 2004). We now show that trastuzumab-resistant and trastuzumab-sensitive cells both surprisingly display trastuzumab-mediated growth inhibition in athymic nude mice. This demonstrates that resistance developed in vitro is not predictive of resistance in vivo. The observation that in vitro resistant cells are sensitive to trastuzumab in vivo could be explained by antibody dependent cellular cytotoxicity (ADCC). Therefore, both parental and trastuzumab-resistant cells were assayed for ADCC in real time on electroplates with and without trastuzumab in the presence of a natural killer cell line (NK-92), and granulocyte or mononuclear cellular fractions isolated from human peripheral blood. Mononuclear cells and NK-92 cells were more effective in killing both parental and trastuzumab-resistant cells in the presence of trastuzumab. Both trastuzumab-resistant cells and trastuzumab-sensitive cells showed similar susceptibility to ADCC despite displaying divergent growth responses to trastuzumab. The granulocyte fraction was able to kill these cells with equal efficacy in the presence or absence of trastuzumab. These results support a model of trastuzumab tumor cell killing in vivo mediated primarily by ADCC from the mononuclear fraction of innate immune cells and suggest that in the clinical setting not only should changes in signaling transduction pathways be studied in acquired tumor resistance to trastuzumab, but also mechanisms by which tumors impede immune function should be evaluated.