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H.R. Lee et al.
Similarly, VCAM-1 is exclusively enriched in subsets for MPC as defined by PDGFRa(+)/Sca-1(+) or PDGFRa(+)/CD51(+) subsets,41-43,50 indicating that the VCAM-1(+) cells that can drive emergence of drug-resistant subsets predominantly overlap with mesenchymal progen- itor cells (Figure 7B).
Supporting this finding, when MN1 leukemic cells were transplanted into the homozygous Bis KO mice, where self- renewing MPC are decreased in the BM,17,51 a significant decrease in the Sca-1(+) subset (LSK) among BM engrafted leukemic cells was observed compared to WT or heterozy- gote Bis KO mice (Figure 7C). This decrease was not associ- ated with altered overall engraftment levels (Figure 7C), con- sistent with the differences between LSK and LK subsets.
Similarly, supporting the role of MPC for the develop- ment of drug resistant leukemic cells, AML patients wo relapsed after treatment exhibited higher numbers of mes- enchymal progenitor subsets (MPC: CD146+/166-)19, 43, 52 in BM, i.e., retrospective studies on AML patients who had undergone relapse within 1 year after complete remission exhibited higher numbers of mesenchymal progenitor sub- sets in the BM than those who maintained complete remis- sion for 5 years (Figure 7D). Notably, this difference was observed regardless of the underlying cytogenetic abnor- mality of the leukemic blasts, indicating that the hetero- geneity of stromal cells could be an additional factor drug- resistance of leukemic cells.
Altogether, this heterogeneity in the BM MPC can influ- ence the stroma-dependent generation of stem cell-like leukemic subsets.
Discussion
Leukemic cell evolution has prevented the effective man- agement of a diverse spectrum of leukemic disease.
Here, using a variety of murine and human leukemia cells both in vitro and in vivo, we show that subsets of leukemic cells can undergo a phenotypic conversion into a stem-like phenotype that exhibit a higher resistance to chemotherapy in the context of stromal contact. This development of chemoresistant subsets by stromal contact was not depend- ent on cell fusion or changes in leukemogenic activities observed in mitochondrial transfer.27,28 The acquisition of the stem cell-like phenotype was reversible, being rapidly reverted to the non-stem cell phenotype under stroma-free conditions independent of difference in cell cycles or apop- tosis (Online Supplementary Figure S10), unlike the stable maintenance of chemoresistance in leukemic clones gener- ated by clonal evolution.
Moreover, the frequencies of LSK cells among leukemic cells in contact with stroma were maintained constant regardless of the phenotype of the initial cell populations. This suggests that the stroma-mediated development of the stem cell-like, drug-resistant subpopulation occurs in a stochastic and reversible manner in leukemic cells with similar probabilities among leukemic cells (equipotent) without clonal predisposition. Reminiscent of these find- ings, recent studies showed that non-stem cancer cells can be spontaneously converted to stem-like state, and these plasticity of cancer cells allows cellular switching between distinct functional states.53,54 Together, these studies raise the possibility that the stochastic development of chemoresistant clones by stromal contact is an intrinsic process of leukemogenesis that could cause a non-
homogenous response to chemotherapy among the leukemic cell populations.
The mechanisms for dynamic equilibrium among differ- ent subsets of leukemic cells remains still unclear. One pos- sibility is a feedback control mechanism that maintains a constant ratio of stem-like versus non-stem-like leukemic cells, probably through cellular interaction between distinct leukemic subsets, as inferred from clonal interactions between heterogenous subsets.55 Similarly, studies on can- cer stem cells have suggested that non-tumorigenic cells regulate the maintenance of cancer stem cells influencing their relative frequencies in the population.56 Since clonal heterogeneity of leukemia or cancer cells underlies the dif- ferential response to chemotherapy and emergence of relapsing clones,55,57,58 the kinetics of generating these stem- like subsets could be a factor for differential response to chemotherapy.
Interestingly, we show that the development of these drug-resistant leukemic subset is facilitated by bi-direction- al cross-talk between stroma and leukemic cells mediated by IL-4, exhibiting resistance to apoptosis (Online Supplementary Figure S11). While IL-4 was implicated in inhibition of leukemic cells and apoptosis,59 we did not find increased apoptosis of the non-stem-like population pre- cluding the selective enrichment of stem-like subsets by IL- 4 (Online Supplementary Figure S12). Moreover, rather than acting directly on the leukemic cells, IL-4 targets stromal cells, which facilitate the generation of LSK subsets. How IL-4 acts on stromal cells to facilitate the generation of a drug-resistant leukemic subset remains unclear. However, we demonstrated a key role for VCAM-1 downstream of IL-4 in MSC leading to tight adherence of leukemic cells and MSC, which was necessary for generation of the LSK subset. Similarly, we found IL-4-dependent induction of gene clusters in MSC whose functions are related to ‘bind- ing function’. This suggests that the mode of interaction between MSC and leukemic cells is altered by IL-4 acting on MSC, which facilitates the development of the drug- resistant leukemic subset.
Interestingly, we also found functional differences between stromal cells in terms of their capacity to drive development of drug-resistant leukemic cells. We found that expression of VCAM-1 in stromal cell was important for adherence-dependent generation of leukemic subsets, while other adhesion molecules we tested did not influence the process (Online Supplementary Figure S13). Importantly, the VCAM-1-expressing stromal cells were selectively enriched in mesenchymal progenitors. Since mesenchymal stromal cells undergo various degenerative changes during leukemia,19,60,61 it is possible that patient-to-patient hetero- geneity in BM mesenchymal progenitor cell content could differentially contribute to the development of drug-resis- tant clones. Consistent with this, AML patients whose BM has higher levels of a primitive (CD146+) subset of mes- enchymal cells19,43,52 that express higher levels of VCAM-1 tend to have a higher risk of leukemic relapse compared to those who maintained complete remission. Thus, inde- pendent of oncogenic mutations or cytogenetic abnormali- ties in the blasts,62 heterogeneity per se in mesenchymal pro- genitors in BM could be another factor for development of drug-resistant leukemic subsets.
In summary, our study reveals an additional mechanism of functional evolution of leukemic cells induced by contact with the mesenchymal stroma that can cause a reversible switch to a stem cell-like, drug-resistant subset independent
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haematologica | 2022; 107(2)