Background: According to the current paradigm, metastases arise from cells of the primary tumor that have intravasated into circulation and carried to distant organs where they extravasate, grow and form new tumors. However, mechanisms underlying the many steps involved in the metastatic cascade remain unexplained. The landmark study by Dr. Isaiah Fidler established that 99% of tumor cells injected IV into mice die within 24 hours of reaching target sites (Fidler, JNCI, 1970). We hypothesized that circulating tumor cells (CTCs) also die upon reaching target organs and release oncogenic cell-free chromatin (cfCh) particles which integrate into their cellular genomes and transform them to form new tumors which masquerade as metastases.

Animal Ethics: The study was approved by Institutional Animal Ethics Committee.

Experiments and Results: 1) MDA-MB-231 human breast cancer and A375 human malignant melanoma cells were dually labelled in their DNA with BrdU and in their histones with CellLight® Histone 2B GFP and injected IV into mice. Immuno-fluorescence (IF) microscopy of lung tissues excised at 48 hours revealed abundant dually labelled cfCh particles in nuclei of lung cells; 2) Unlabeled MDA-MB-231 and A375 cells were similarly injected, and IF analysis of lung tissues excised at 72 hours revealed 5 - 20 fold increase in all ten hallmarks of cancer defined by Hanahan and Weinberg when compared with un-injected control lung tissues; 3) Mice injected with unlabeled cells were followed for 2-3 months until they developed lung metastasis which were excised and found to contain both mouse and human DNA when examined by highly specific FISH probes. The tumor cells also contained human specific HLA ABC Class I and mouse specific MHC Class II proteins when examined by highly specific mAbs; 4) Metaphase spreads prepared from primary cultures from lung metastasis were found to contain chimeric chromosomes with both mouse and human DNA. The cells expressed both human and mouse specific proteins in the same cells; 5) Several single cell clones developed from primary cultures were also found to contain chimeric chromosomes of mouse and human DNA and to express both mouse and human specific proteins; 6) cfCh particles were isolated from Adriamycin treated dying MDA-MB-231 cells by modification of a method described by us (Mittra et al J Biosciences 2015). When the isolated cfCh particles were injected IV into mice, they went on to develop lung metastasis after ˜ 7 months, thereby directly implicating cfCh in development of metastases. The cfCh induced tumors were again found to contain both mouse and human specific DNA and proteins.

Conclusion: Metastases arise as new cancers from cells of target organs transformed by cfCh particles released from dying CTCs. This conclusion challenges the dogma that metastases arise from cells that are derived from the primary tumor.

Citation Format: Indraneel Mittra, Venkata Raghuram Gorantla, Soniya Shende, Naveen Kumar Khare, Harshada Wani, Ratnam Prasad, Kavita Pal. Metastases arise as new cancers from cells of target organs transformed by cell-free chromatin particles released from dying circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2875.