gms | German Medical Science

Objective: The aim of this study is to evaluate the effects of cancer immunoediting on the clonality of metastatic tumors in distinctive locations. Our hypothesis is that the peripheral immune system favors the proliferation of some specific metastasis subclones, however such subpopulations have not yet been identified.

Methods: Murine melanoma (B16), NSCLC (LLC) and breast cancer (EO771) cell lines were transduced with lentivirus carrying either mCherry (Red) Venus (Green) or Cerulean (Blue) fluorescent proteins (RGB) and selected until a stable expression was achieved. Syngeneic tumor cells were injected subcutaneously, intracardially or stereotactically implanted in the striatum of C57Bl/6 immunocompetent mice. Four specific organs (lung, liver, brain, adrenal glands) and any additional macroscopic metastases were analyzed by flow-cytometry and immunohistochemistry to investigate tumor heterogeneity and clonal selection patterns of metastatic tumor cells. The degree of clonal restriction was assessed by quantification of the chromaticity of RGB labeled tumor cells analyzed by flow cytometry.

Results: Overall 64 mice were analyzed (B16 n=21; LLC n=21; EO 771 n=22). Survival was significantly altered by type of tumor cell as well as type of injection method utilized, with intracerebrally injected EO771-RGB breast cancer cells yielding the shortest survival (median 10 d, p<0.0001, Log-rank test). Metastasis spread also differs based on cell line and injection location with the intracardiac B16 injection leading to the highest number of affected organs (12/12 organs). Interestingly, intracranial injection of these non-glioma tumor cell lines also led to metastatic spread, although to a lesser extent. In the primary site of injections we did not observe any restriction in our color barcode, meaning that most cells injected were able to grow without selection from the microenvironment. However, in the metastases, particularly in the adrenal gland, an important clonal selection occurred leading us to conclude that only very specific clones were able to escape the primary site and proliferate into a new environment. Interestingly, these clones seem to differ between mice.

Conclusion: The confirmation that different clones can emerge from the primary site to colonize specific organs raised the idea that immunoediting on tumor cells is a major driver of tumor heterogeneity that requires a deeper understanding and multiple site sampling to enable new therapeutic options.