Residual disease on a dish: A practical approach to deep intrinsic resistance in melanoma

Cancer is an evolution like process; wherein genetically abnormal cells persist despite a variety of mechanisms designed to kill such cells. Immune checkpoint and targeted therapies have significantly improved outcomes in melanoma. However, residual disease remaining after treatment, as indicated by a lack of complete pathological response or as detected by other sensitive methods, often evolves further leading to recurrence/metastasis. Thus, there is a need to inhibit or eradicate residual disease to prevent recurrence. In this regard, there is a lack of good preclinical models of residual disease, particularly for testing new therapies that would inhibit quiescent or slow-growing melanoma cells. This is important since opportunistically switching to quiescence and to survive in deep quiescence are major hurdles in improving outcomes. Our approach to a cell culture model of residual disease aligns with the idea that selection pressures are important in enforcing cancer cell quiescence. A selection pressure involving a severe and extended metabolic change, such as a lack of glutamine in culture medium, is an excellent choice for eliminating bulk of sensitive cells that proliferate in artificially rich medium. We subjected two highly metastatic melanoma cell lines, A375SM (representing undifferentiated melanoma with a common BRAF V600E mutation and homozygous CDKN2A deletion) and B16-BL6 (suitable for syngeneic mouse model), to glutamine deficiency in two ways: 1) culture in Gln-free medium (with low 27 µM Gln coming from serum, instead of 4 mM Gln in complete medium), and 2) culture in stringent Gln-free medium (near zero Gln with the use of dialyzed serum in medium). More than 99% cells died in Gln-free medium; rare cells survived in deep quiescence, and then gradually exited quiescence after 4-5 weeks and proliferated indefinitely. We extended the quiescence to 7 weeks and beyond by culturing A375SM cells in stringent Gln-free medium. In these experiments, we replaced medium with fresh medium every 2-3 weeks. Interestingly, if we did not replace medium for an extended period (50 days), we found that A375SM cells did not exit quiescence. We interpret that rare cells surviving in deep quiescence gradually develop an ability to exit quiescence; however, this exit or/and proliferation requires some glutamine. By not changing medium for an extended period, low level of Gln got exhausted, and cells stayed quiescent. We refer to the cells selected in this manner as metabolically adaptable (MA); they are broadly adaptable. The MA cells were much more resistant to paclitaxel than were their parental cell lines. Our results support the feasibility of the approach for modeling residual disease. In conclusion, when we approach deep intrinsic resistance in melanoma with a new perspective (abnormal quiescence besides abnormal proliferation leading to recurrence), new insights and fresh solutions are likely to emerge.